SSTR2-based Reporters for Assessing Gene Transfer into Non-Small Cell Lung Cancer: Evaluation using an Intrathoracic Mouse Model.

Hum Gene Ther. 2010 Jul 23;

Authors: Singh SP, Han L, Ravoori MK, Solis LM, Roth JA, Ji L, Wistuba II, Kundra V

Purpose: The most common cause of cancer-related deaths in North America is lung cancer, of which, 85% is due to non-small cell (NSCLC). Gene therapy is a promising approach, but has been hindered by lack of methods for localizing and quantifying gene expression in vivo. Human somatostatin receptor subtype-2 (SSTR2)-based reporters can be used to follow gene expression in vivo using ligands with greater affinity for this subtype. NSCLC's can express somatostatin receptor subtypes, which may interfere with SSTR2-based reporters. We assessed whether a SSTR2-based reporter can serve as reporter of gene transfer into NSCLC's. Material and Methods: SSTR subtype expression was assessed in NSCLC cell lines A549, H460, and H1299 using RT-PCR. After infection with an adenovirus containing hemagglutinin-A-tagged-SSTR2 (Ad-HA-SSTR2) or control insert, expression was assessed by immunologic techniques and binding to clinically-approved 111In-octreotide. In vivo, after MR imaging, intrathoracic H460 tumors were injected with Ad-HA-SSTR2 or control virus (n=6 mice/group) under ultrasound guidance. Intravenous injection of 111In-octreotide two days later was followed by planar and SPECT imaging. Biodistribution into tumors was assessed in vivo using anatomic MR and functional gamma-camera images and ex vivo using excised organs/tumors. In human lung tumor samples (n=70), SSTR2 expression was assessed using immunohistochemistry. Results: All three NSCLC cell lines expressed different SSTR subtypes, but none expressed SSTR2. Upon Ad-HA-SSTR2 infection, HA-SSTR2 expression was seen in all three cell lines using antibodies targeting the HA domain or 111In-octreotide targeting the receptor domain (P<.05). Intrathoracic tumors infected with Ad-HA-SSTR2 were clearly visible by gamma camera imaging; expression was quantified by both in vivo and ex vivo biodistribution analysis and demonstrated greater uptake in tumors infected with Ad-HA-SSTR2 compared to control virus (P<.05). Immunohistochemistry found that 78% of NSCLC's are negative for and 13% have low levels of SSTR subtype 2 expression. Conclusion: SSTR2-based reporters can serve as reporters of gene transfer into non-small cell lung cancers.

PMID: 20653396 [PubMed - as supplied by publisher]

Enhancement of antitumor vaccine in ablated hepatocellular carcinoma by high-intensity focused ultrasound.

World J Gastroenterol. 2010 Jul 28;16(28):3584-91

Authors: Zhang Y, Deng J, Feng J, Wu F

AIM: To investigate whether tumor debris created by high-intensity focused ultrasound (HIFU) could trigger antitumor immunity in a mouse hepatocellular carcinoma model. METHODS: Twenty C57BL/6J mice bearing H22 hepatocellular carcinoma were used to generate antitumor vaccines. Ten mice underwent HIFU ablation, and the remaining 10 mice received a sham-HIFU procedure with no ultrasound irradiation. Sixty normal mice were randomly divided into HIFU vaccine, tumor vaccine and control groups. These mice were immunized with HIFU-generated vaccine, tumor-generated vaccine, and saline, respectively. In addition, 20 mice bearing H22 tumors were successfully treated with HIFU ablation. The protective immunity of the vaccinated mice was investigated before and after a subsequent H22 tumor challenge. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the cytotoxicity of splenic lymphocytes co-cultured with H22 cells was determined in vitro before the tumor challenge, and tumor volume and survival were measured in vivo after the challenge in each group. The mechanism was also explored by loading the vaccines with bone marrow-derived dendritic cells (DCs). RESULTS: Compared to the control, HIFU therapy, tumor-generated and HIFU-generated vaccines significantly increased cytolytic activity against H22 cells in the splenocytes of the vaccinated mice (P < 0.001). The tumor volume was significantly smaller in the HIFU vaccine group than in the tumor vaccine group (P < 0.05) and control group (P < 0.01). However, there was no tumor growth after H22 rechallenge in the HIFU therapy group. Forty-eight-day survival rate was 100% in mice in the HIFU therapy group, 30% in both the HIFU vaccine and tumor vaccine groups, and 20% in the control group, indicating that the HIFU-treated mice displayed significantly longer survival than the vaccinated mice in the remaining three groups (P < 0.001). After bone marrow-derived DCs were incubated with HIFU-generated and tumor-generated vaccines, the number of mature DCs expressing MHC-II(+), CD80(+) and CD86(+) molecules was significantly increased, and interleukin-12 and interferon-gamma levels were significantly higher in the supernatants when compared with immature DCs incubated with mouse serum (P < 0.001). However, no differences of the number of mature DCs and cytokine levels were observed between the HIFU-generated and tumor-generated vaccines (P > 0.05). CONCLUSION: Tumor debris remaining after HIFU can improve tumor immunogenicity. This debris releases tumor antigens as an effective vaccine to develop host antitumor immune response after HIFU ablation.

PMID: 20653069 [PubMed - in process]

A microcomposite hydrogel for repeated on-demand ultrasound-triggered drug delivery.

Biomaterials. 2010 Jul;31(19):5208-17

Authors: Epstein-Barash H, Orbey G, Polat BE, Ewoldt RH, Feshitan J, Langer R, Borden MA, Kohane DS

Here we develop an injectable composite system based for repeated ultrasound-triggered on-demand drug delivery. An in situ-cross-linking hydrogel maintains model drug (dye)-containing liposomes in close proximity to gas-filled microbubbles that serve to enhance release events induced by ultrasound application. Dye release is tunable by varying the proportions of the liposomal and microbubble components, as well as the duration and intensity of the ultrasound pulses in vitro. Dye is minimal at baseline. The composite shows minimal cytotoxicity in vitro, and benign tissue reaction after subcutaneous injection in rats. Ultrasound application also triggers drug release for two weeks after injection in vivo.

PMID: 20347484 [PubMed - indexed for MEDLINE]

In vivo photoacoustic imaging of transverse blood flow by using Doppler broadening of bandwidth.

Opt Lett. 2010 May 1;35(9):1419-21

Authors: Yao J, Maslov KI, Shi Y, Taber LA, Wang LV

A method is proposed to measure transverse blood flow by using photoacoustic Doppler broadening of bandwidth. By measuring bovine blood flowing through a plastic tube, the linear dependence of the broadening on the flow speed was validated. The blood flow of the microvasculature in a mouse ear and a chicken embryo (stage 16) was also studied.

PMID: 20436589 [PubMed - indexed for MEDLINE]

A high-frequency, high frame rate duplex ultrasound linear array imaging system for small animal imaging.

IEEE Trans Ultrason Ferroelectr Freq Control. 2010 Jul;57(7):1548-57

Authors: Zhang L, Xu X, Hu C, Sun L, Yen J, Cannata J, Shung K

High-frequency (HF) ultrasound imaging has been shown to be useful for non-invasively imaging anatomical structures of the eye and small animals in biological and pharmaceutical research, achieving superior spatial resolution. Cardiovascular research utilizing mice requires not only realtime B-scan imaging, but also ultrasound Doppler to evaluate both anatomy and blood flow of the mouse heart. This paper reports the development of an HF ultrasound duplex imaging system capable of both B-mode imaging and Doppler flow measurements, using a 64-element linear array. The system included an HF pulsed-wave Doppler module, a 32-channel HF B-mode imaging module, a PC with a 200 MS/s 14-bit A/D card, and real-time LabView software. A 50 dB SNR and a depth of penetration of larger than 12 mm were achieved using a 35-MHz linear array with 50 ¿ m pitch. The two-way beam widths were determined to be 165 to 260 ¿m and the clutterenergy¿ to¿total-energy ratio (CTR) were 9.1 to 12 dB when the array was electronically focused at different focal points at depths from 4.8 to 9.6 mm. The system is capable of acquiring real-time B-mode images at a rate greater than 400 frames per second (fps) for a 4.8 x 13 mm field of view, using a 30- MHz 64-element linear array with 100 mum pitch. Sample in vivo cardiac high frame rate images and duplex images of mouse hearts are shown to assess its current imaging capability and performance for small animals.

PMID: 20639149 [PubMed - in process]

Video rate optoacoustic tomography of mouse kidney perfusion.

Opt Lett. 2010 Jul 15;35(14):2475-7

Authors: Buehler A, Herzog E, Razansky D, Ntziachristos V

Optoacoustic tomography can visualize optical contrast in tissues while capitalizing on the advantages of ultrasound, such as high spatial resolution and fast imaging capabilities. We report a novel multispectral optoacoustic tomography system for deep tissue small animal imaging. The previously undocumented capacity of whole-body optoacoustic tomography at a video rate has been demonstrated by visualizing mouse kidney perfusion using Indocyanine Green in vivo.

PMID: 20634868 [PubMed - in process]

Cardiovascular phenotyping of the mouse heart using a 4D radial acquisition and liposomal Gd-DTPA-BMA.

Magn Reson Med. 2010 Apr;63(4):979-87

Authors: Bucholz E, Ghaghada K, Qi Y, Mukundan S, Rockman HA, Johnson GA

MR microscopy has enormous potential for small-animal cardiac imaging because it is capable of producing volumetric images at multiple time points to accurately measure cardiac function. MR has not been used as frequently as ultrasound to measure cardiac function in the small animal because the MR methods required relatively long scan times, limiting throughput. Here, we demonstrate four-dimensional radial acquisition in conjunction with a liposomal blood pool agent to explore functional differences in three populations of mice: six C57BL/6J mice, six DBA/2J mice, and six DBA/2J CSQ+ mice, all with the same gestational age and approximately the same weight. Cardiovascular function was determined by measuring both left ventricular and right ventricular end diastolic volume, end systolic volume, stroke volume, and ejection fraction. Statistical significance was observed in end diastolic volume, end systolic volume, and ejection fraction for left ventricular measurements between all three populations of mice. No statistically significant difference was observed in stroke volume in either the left or right ventricle for any of the three populations of mice. This study shows that MRI is capable of efficient, high-throughput, four-dimensional cardiovascular phenotyping of the mouse.

PMID: 20373399 [PubMed - indexed for MEDLINE]

Low-intensity ultrasound stimulation prevents osteoporotic bone loss in young adult ovariectomized mice.

J Orthop Res. 2010 Jul 6;

Authors: Lim D, Ko CY, Seo DH, Woo DG, Kim JM, Chun KJ, Kim HS

Osteoporosis is a disease characterized by low bone mass, increased bone fragility, and a greater risk for bone fracture. Currently, pharmacological intervention can generally aid in the prevention and treatment of osteoporosis, but these therapies are often accompanied by undesirable side effects. Therefore, alternative therapies that minimize side effects are necessary. Biophysical stimuli, especially low-intensity ultrasound stimulation (LIUS), may be potential alternatives to drug-based therapies for osteoporosis. Hence, we sought to address whether LIUS therapy can effectively prevent or treat osteoporotic bone loss induced by estrogen deficiency. LIUS (1.5 MHz frequency, 1.0 kHz pulse repetition on frequency, 30 mW/cm(2) intensity, 200 micros pulse length) was applied to right tibiae of eight 14-week-old ovariectomized virgin ICR female mice for 20 min per day, 5 days per week, over a 6-week period. Changes in 3D structural bone characteristics were detected using in vivo micro-computed tomography. Left tibiae served as controls. Structural characteristics including bone volume/tissue volume, trabecular number, trabecular bone pattern factor, and mean polar moment inertia were significantly enhanced 6 weeks after LIUS compared to the control, nonstimulated group (p < 0.05). In particular, the bone volume/tissue volume in the region exposed directly to LIUS was significantly higher in the treated group (p < 0.05). These findings indicate that new bone formation may be activated or that bone structure may be maintained by LIUS, and that LIUS may be effective for preventing estrogen deficiency-induced bone loss. (c) 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

PMID: 20607839 [PubMed - as supplied by publisher]

Xenografted human amniotic membrane-derived mesenchymal stem cells are immunologically tolerated and transdifferentiated into cardiomyocytes.

Circ Res. 2010 May 28;106(10):1613-23

Authors: Tsuji H, Miyoshi S, Ikegami Y, Hida N, Asada H, Togashi I, Suzuki J, Satake M, Nakamizo H, Tanaka M, Mori T, Segawa K, Nishiyama N, Inoue J, Makino H, Miyado K, Ogawa S, Yoshimura Y, Umezawa A

RATIONALE: Amniotic membrane is known to have the ability to transdifferentiate into multiple organs and is expected to stimulate a reduced immunologic reaction. OBJECTIVE: Determine whether human amniotic membrane-derived mesenchymal cells (hAMCs) can be an ideal allograftable stem cell source for cardiac regenerative medicine. METHODS AND RESULTS: We established hAMCs. After cardiomyogenic induction in vitro, hAMCs beat spontaneously, and the calculated cardiomyogenic transdifferentiation efficiency was 33%. Transplantation of hAMCs 2 weeks after myocardial infarction improved impaired left ventricular fractional shortening measured by echocardiogram (34+/-2% [n=8] to 39+/-2% [n=11]; P<0.05) and decreased myocardial fibrosis area (18+/-1% [n=9] to 13+/-1% [n=10]; P<0.05), significantly. Furthermore hAMCs transplanted into the infarcted myocardium of Wistar rats were transdifferentiated into cardiomyocytes in situ and survived for more than 4 weeks after the transplantation without using any immunosuppressant. Immunologic tolerance was caused by the hAMC-derived HLA-G expression, lack of MHC expression of hAMCs, and activation of FOXP3-positive regulatory T cells. Administration of IL-10 or progesterone, which is known to play an important role in feto-maternal tolerance during pregnancy, markedly increased HLA-G expression in hAMCs in vitro and, surprisingly, also increased cardiomyogenic transdifferentiation efficiency in vitro and in vivo. CONCLUSIONS: Because hAMCs have a high ability to transdifferentiate into cardiomyocytes and to acquire immunologic tolerance in vivo, they can be a promising cellular source for allograftable stem cells for cardiac regenerative medicine.

PMID: 20508201 [PubMed - indexed for MEDLINE]

Radiosensitization and Stromal Imaging Response Correlates for the HIF-1 Inhibitor PX-478 Given with or without Chemotherapy in Pancreatic Cancer.

Mol Cancer Ther. 2010 Jun 29;

Authors: Schwartz DL, Bankson JA, Lemos R, Lai SY, Thittai AK, He Y, Hostetter G, Demeure MJ, Von Hoff DD, Powis G

Growing tumors are hypoxic and respond to microenvironmental stress through increased expression of the hypoxia inducible factor-1alpha (HIF-1alpha) transcription factor, resulting in an adaptive switch to glycolytic metabolism, angiogenic signaling, survival, and metastasis. HIF-1alpha expression is associated with tumor resistance to cytotoxic therapy and inferior patient outcomes. Pancreatic cancer is the most hypoxic of all solid tumors and remains refractory to current chemoradiotherapy. We have seen nuclear HIF-1alpha in 88% of human pancreatic ductal carcinoma but in only 16% of normal pancreas. Stroma adjacent to the pancreatic ductal carcinoma also showed HIF-1alpha in 43% of cases. We investigated the novel selective HIF-1alpha inhibitor PX-478 on in vitro and in vivo radiation response of human pancreatic cancer models. Inhibition of HIF-1alpha by PX-478 increased cell killing by radiation. In mice with Panc-1, CF-PAC-1, or SU.86.86 pancreatic xenografts, concurrent administration of PX-478 potentiated the antitumor effects of fractionated radiation, with or without combined treatment with 5-fluorouracil or gemcitabine. Alternative sequencing of PX-478 with fractionated radiotherapy suggests optimal radiosensitization with concurrent or neoadjuvant administration of drug. Early tumor responses to combined PX-478/radiation treatment could be rapidly and repeatedly quantified by vascular imaging biomarkers. Dual-tracer dynamic contrast enhanced-magnetic resonance imaging and ultrasound imaging discriminated response to combined treatment prior to detection of differences in anatomic tumor size at 10 days posttreatment. Therefore, PX-478 is a mechanistically appealing and potentially clinically relevant enhancer of pancreatic cancer radiosensitivity, inhibiting tumor and stromal HIF-1 proangiogenic signaling and reducing the innate radiation resistance of hypoxic tumor cells. Mol Cancer Ther; 9(7); 2057-67. (c)2010 AACR.

PMID: 20587661 [PubMed - as supplied by publisher]

Both the transplantation of somatic cell nuclear transfer- and fertilization-derived mouse embryonic stem cells with temperature-responsive chitosan hydrogel improve myocardial performance in infarcted rat hearts.

Tissue Eng Part A. 2010 Apr;16(4):1303-15

Authors: Lü S, Wang H, Lu W, Liu S, Lin Q, Li D, Duan C, Hao T, Zhou J, Wang Y, Gao S, Wang C

The transplantation of embryonic stem cells could improve cardiac function but was limited by immune rejection as well as low cell retention and survival within the ischemic tissues. The somatic cell nuclear transfer (SCNT) is practical to generate autologous histocompatible stem (nuclear-transferred embryonic stem [NTES]) cells for diseases, but NTES may be arguably unsafe for therapeutic application. The temperature-responsive chitosan hydrogel is a suitable matrix in cell transplantation. As the scaffold, chitosan hydrogel was coinjected with NTES cells into the left ventricular wall of rat infarction models. Detailed histological analysis and echocardiography were performed to determine the structure and functional consequences of transplantation. The myocardial performance in SCNT- and fertilization-derived mouse ES cell transplantation with chitosan hydrogel was also compared. The results showed that both the 24-h cell retention and 4-week graft size were significantly greater in the NTES + chitosan group than that of NTES + phosphate-buffered saline (PBS) group (p < 0.01). The NTES cells might differentiate into cardiomyocytes in vivo. The heart function improved significantly in the chitosan + NTES group (fractional shortening: 28.7% +/- 2.8%) compared with that of PBS + NTES group (fractional shortening: 25.2% +/- 2.9%) at 4 weeks after transplantation (p < 0.01). In addition, the arteriole/venule densities within the infarcted area improved significantly in the chitosan + NTES group (280 +/- 17/mm(2)) compared with that of PBS + NTES group (234 +/- 16/mm(2)) at 4 weeks after transplantation (p < 0.01). There was no difference in the myocardial performance in SCNT- and fertilization-derived mouse ES cell transplantation with chitosan hydrogel. The NTES cells with chitosan hydrogel have been proved to possess therapeutic potential to improve the function of infarcted heart. Thus the method of in situ injectable tissue engineering is promising clinically.

PMID: 19905874 [PubMed - indexed for MEDLINE]

Distinctive ERK and p38 signaling in remote and infarcted myocardium during post-MI remodeling in the mouse.

J Cell Biochem. 2010 Apr 15;109(6):1185-91

Authors: Yeh CC, Li H, Malhotra D, Turcato S, Nicholas S, Tu R, Zhu BQ, Cha J, Swigart PM, Myagmar BE, Baker AJ, Simpson PC, Mann MJ

Global activation of MAP kinases has been reported in both human and experimental heart failure. Chronic remodeling of the surviving ventricular wall after myocardial infarction (MI) involves both myocyte loss and fibrosis; we hypothesized that this cardiomyopathy involves differential shifts in pro- and anti-apoptotic MAP kinase signaling in cardiac myocyte (CM) and non-myocyte. Cardiomyopathy after coronary artery ligation in mice was characterized by echocardiography, ex vivo Langendorff preparation, histologic analysis and measurements of apoptosis. Phosphorylation (activation) of signaling molecules was analyzed by Western blot, ELISA and immunohistochemistry. Post-MI remodeling involved dramatic changes in the phosphorylation of both stress-activated MAP (SAP) kinase p38 as well as ERK, a known mediator of cell survival, but not of SAP kinase JNK or the anti-apoptotic mediator of PI3K, Akt. Phosphorylation of p38 rose early after MI in the infarct, whereas a more gradual rise in the remote myocardium accompanied a rise in apoptosis in that region. In both areas, ERK phosphorylation was lowest early after MI and rose steadily thereafter, though infarct phosphorylation was consistently higher. Immunostaining of p-ERK localized to fibrotic areas populated primarily by non-myocytes, whereas staining of p38 phosphorylation was stronger in areas of progressive CM apoptosis. Relative segregation of CMs and non-myocytes in different regions of the post-MI myocardium revealed signaling patterns that imply cell type-specific changes in pro- and anti-apoptotic MAP kinase signaling. Prevention of myocyte loss and of LV remodeling after MI may therefore require cell type-specific manipulation of p38 and ERK activation.

PMID: 20186881 [PubMed - indexed for MEDLINE]

Breviscapine protects against cardiac hypertrophy through blocking PKC-alpha-dependent signaling.

J Cell Biochem. 2010 Apr 15;109(6):1158-71

Authors: Yan L, Huang H, Tang QZ, Zhu LH, Wang L, Liu C, Bian ZY, Li H

Breviscapine is a mixture of flavonoid glycosides extracted from the Chinese herbs. Previous studies have shown that breviscapine possesses comprehensive pharmacological functions. However, very little is known about whether breviscapine have protective role on cardiac hypertrophy. The aim of the present study was to determine whether breviscapine attenuates cardiac hypertrophy induced by angiotensin II (Ang II) in cultured neonatal rat cardiac myocytes in vitro and pressure-overload-induced cardiac hypertrophy in mice in vivo. Our data demonstrated that breviscapine (2.5-15 microM) dose-dependently blocked cardiac hypertrophy induced by Ang II (1 microM) in vitro. The results further revealed that breviscapine (50 mg/kg/day) prevented cardiac hypertrophy induced by aortic banding as assessed by heart weight/body weight and lung weight/body weight ratios, echocardiographic parameters, and gene expression of hypertrophic markers. The inhibitory effect of breviscapine on cardiac hypertrophy is mediated by disrupting PKC-alpha-dependent ERK1/2 and PI3K/AKT signaling. Further studies showed that breviscapine inhibited inflammation by blocking NF-kappaB signaling, and attenuated fibrosis and collagen synthesis through abrogating Smad2/3 signaling. Therefore, these findings indicate that breviscapine, which is a potentially safe and inexpensive therapy for clinical use, has protective potential in targeting cardiac hypertrophy and fibrosis through suppression of PKC-alpha-dependent signaling.

PMID: 20127712 [PubMed - indexed for MEDLINE]

Contrast Ultrasonography: Necessity of Linear Data Processing for the Quantification of Tumor Vascularization.

Ultraschall Med. 2010 Jun 24;

Authors: Peronneau P, Lassau N, Leguerney I, Roche A, Cosgrove D

PURPOSE: This study is intended to compare the value of uncompressed ultrasonic data, obtained after linear power detection of the ultrasonic radiofrequencies that we call linear data, with usual compressed video data for the quantification of tumor perfusion, particularly for monitoring antivascular therapy. MATERIALS AND METHODS: To form a clinically useful ultrasonic image, the detected power of the received signals (linear data) is compressed in a quasi-logarithmic fashion in order to match the limited dynamic range of the video monitor. The resulting reduced range of signals from an injected contrast agent may limit the sensitivity to changes in the time-intensity curves. Following a theoretical evaluation of the effects of compression on time-intensity curves and as an in vivo example, we measured at different times the effects of an antivascular drug administered to mice bearing melanoma tumors. The mean time-intensity curves within the tumors after bolus injection of a contrast agent were determined using both linear and video data. Linearized data was recovered using the inverse of the true scanner's compression law, which was experimentally determined. Three features were extracted from the time-intensity curves: peak intensity (PI), time to peak intensity (TPI) and area under the curve in the wash-in phase (AUC (wash-in)). When contrast reached its maximum value, the coefficient of variation reflecting the heterogeneity of the intensity of contrast uptake within the tumor, was computed using both data sets. RESULTS: TPI was found to be similar with either data set (r = 0.98, p < 0.05, factor of 1.09). Linear PI and AUC (wash-in) had significantly earlier decreases after drug administration than video data (p = 0.015 and p = 0.03, respectively). The coefficient of variation was significantly lower when using video rather than linear data (p < 10 (-4)). CONCLUSION: In conclusion, the use of linear data is the only mathematically valid methodology for determining a tumor's time-intensity curve and, in practice, it allows earlier demonstration of responses to antivascular drugs.

PMID: 20577941 [PubMed - as supplied by publisher]

Treatment of heart failure by a methanocarba derivative of adenosine monophosphate: implication for a role of cardiac purinergic P2X receptors.

J Pharmacol Exp Ther. 2010 Jun;333(3):920-8

Authors: Zhou SY, Mamdani M, Qanud K, Shen JB, Pappano AJ, Kumar TS, Jacobson KA, Hintze T, Recchia FA, Liang BT

Evidence is accumulating to support a potentially important role for purinergic (P2X) receptors in heart failure (HF). We tested the hypothesis that a hydrolysis-resistant nucleotide analog with agonist activity at myocardial P2X receptors (P2XRs) improves the systolic HF phenotype in mouse and dog models. We developed a hydrolysis-resistant adenosine monophosphate derivative, (1'S,2R,3S,4'R,5'S)-4-(6-amino-2-chloro-9H-purin-9-yl)-1-[phosphoryloxymethyl] bicycle[3.1.0]hexane-2,3-diol) (MRS2339), with agonist activity at native cardiac P2XRs. Chronic MRS2339 infusion in postinfarct and calsequestrin (CSQ) mice with HF resulted in higher rates of pressure change (+dP/dt), left ventricle (LV)-developed pressure, and cardiac output in an in vitro working heart model. Heart function in vivo, as determined by echocardiography-derived fractional shortening, was also improved in MRS2339-infused mice. The beneficial effect of MRS2339 was dose-dependent and was identical to that produced by cardiac myocyte-specific overexpression of the P2X(4) receptor. The HF improvement was associated with the preservation of LV wall thickness in both systole and diastole in postinfarct and CSQ mice. In dogs with pacing-induced HF, MRS2339 infusion reduced left ventricular end-diastolic pressure, improved arterial oxygenation, and increased +dP/dt. MRS2339 treatment also decreased LV chamber size in mice and dogs with HF. In murine and canine models of systolic HF, in vivo administration of a P2X nucleotide agonist improved contractile function and cardiac performance. These actions were associated with preserved LV wall thickness and decreased LV remodeling. The data are consistent with a role of cardiac P2XRs in mediating the beneficial effect of this agonist.

PMID: 20200116 [PubMed - indexed for MEDLINE]

Transthoracic echocardiography in mice.

J Vis Exp. 2010;(39):

Authors: Respress JL, Wehrens XH

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene expression. Transgenic and gene targeting methods can be used to generate mice with altered cardiac size and function, and as a result, in vivo techniques are needed to evaluate their cardiac phenotype. Transthoracic echocardiography, pulse wave Doppler (PWD), and tissue Doppler imaging (TDI) can be used to provide dimensional measurements of the mouse heart and to quantify the degree of cardiac systolic and diastolic performance. Two-dimensional imaging is used to detect abnormal anatomy or movements of the left ventricle, whereas M-mode echo is used for quantification of cardiac dimensions and contractility. In addition, PWD is used to quantify localized velocity of turbulent flow, whereas TDI is used to measure the velocity of myocardial motion. Thus, transthoracic echocardiography offers a comprehensive method for the noninvasive evaluation of cardiac function in mice.

PMID: 20517201 [PubMed - indexed for MEDLINE]

Rapid functional dissection of genetic networks via tissue-specific transduction and RNAi in mouse embryos.

Nat Med. 2010 Jun 6;

Authors: Beronja S, Livshits G, Williams S, Fuchs E

Using ultrasound-guided in utero infections of fluorescently traceable lentiviruses carrying RNAi or Cre recombinase into mouse embryos, we have demonstrated noninvasive, highly efficient selective transduction of surface epithelium, in which progenitors stably incorporate and propagate the desired genetic alterations. We achieved epidermal-specific infection using small generic promoters of existing lentiviral short hairpin RNA libraries, thus enabling rapid assessment of gene function as well as complex genetic interactions in skin morphogenesis and disease in vivo. We adapted this technology to devise a new quantitative method for ascertaining whether a gene confers a growth advantage or disadvantage in skin tumorigenesis. Using alpha1-catenin as a model, we uncover new insights into its role as a widely expressed tumor suppressor and reveal physiological interactions between Ctnna1 and the Hras1-Mapk3 and Trp53 gene pathways in regulating skin cell proliferation and apoptosis. Our study illustrates the strategy and its broad applicability for investigations of tissue morphogenesis, lineage specification and cancers.

PMID: 20526348 [PubMed - as supplied by publisher]

Ultrasound dose fractionation in sonodynamic therapy.

Ultrasound Med Biol. 2010 Jun;36(6):880-7

Authors: Barati AH, Mokhtari-Dizaji M

Sonodynamic therapy (SDT) is a new modality in cancer therapy and it is based on preferential uptake and/or retention of a sonosensitizing drug in tumor tissues and subsequent activation of the drug by ultrasound. The dose fractionation effect in radiation therapy has been known for more than a century, but it is not reported in SDT so far. In this study, the in vivo antitumor effect of the simultaneous dual-frequency ultrasound (1 MHz and 150 kHz) at low-level intensity (cumulative I(SATA) = 2.2W/cm(2); total energy density 3960J/cm(2); for 30 min sonication) in combination with the sonosensitizer of photofrin (PF) (sodium porfimer) was investigated in dose fractionation regime in a spontaneous murine model of breast adenocarcinoma in Balb/c mice. The tumor-bearing mice were divided into six groups (n = 8 to 10): Untreated groups included control and sham; experimental groups were treated with 5 mg/kg intravenous injection of PF alone, with combined PF and ultrasound for 30-min sonication in one fraction at 24 h after PF administration; with combined PF and ultrasound for 30 min sonicatin in three fraction at 18, 24 and 30 h after PF administration; and finally with combined PF and ultrasound for 30 min sonication in five fraction at 12, 18, 24, 30 and 36 h after PF administration. The tumor growth delay (TGD) parameters and the percent of apoptotic index AI (%) were measured in treated and untreated groups. The results show that the TGD parameters in treatment groups with combined drug and ultrasound fractionation mode were significantly different compared with other groups (p < 0.05). Also the sonodynamic ultrasound dose fractionation in five fractions is more effective than of the three-fraction regime. The AI of the tumor tissues treated by ultrasound dose fractionation was also significantly higher in the other groups (p < 0.05), in which the AI (%) in the group treated with five fractions was higher with respect to group treated with 3 fractions (11.56 +/- 1.2; 8.7 +/- 0.87), respectively. In conclusion, the ultrasound dose fractionation can be useful in therapeutic effect in SDT and may have future clinical applications.

PMID: 20510185 [PubMed - in process]

Ultrasound-assisted non-viral gene transfer to the salivary glands.

Gene Ther. 2010 May 27;

Authors: Passineau MJ, Zourelias L, Machen L, Edwards PC, Benza RL

We report a non-viral gene transfer method using ultrasound induced microbubble destruction to allow the uptake of plasmid gene transfer vectors to the cells of the mouse salivary gland. The Luciferase (Luc) reporter gene, driven by a cytomegalovirus (CMV) promoter, was delivered unilaterally to the submandibular salivary gland via retroductal cannulation and Luc expression was monitored with in vivo imaging. The CMV-Luc plasmid was delivered to the salivary gland in a carrier solution containing microbubbles composed of lipid-encased perfluoropropane gas, with two different concentrations of microbubbles used (100 and 15% volume/volume). An Adenoviral (Ad) vector using an identical CMV-Luc expression cassette was used as a positive control at two different dosages. Whereas ultrasound-assisted gene transfer (UAGT) with 100% microbubbles was weak and rapidly extinguished, UAGT with the 15% microbubble solution was robust and stable for 28 days. UAGT seems to be a practicable and promising method for non-viral gene delivery to the salivary glands.Gene Therapy advance online publication, 27 May 2010; doi:10.1038/gt.2010.86.

PMID: 20508599 [PubMed - as supplied by publisher]

Sonoporation mediated immunogene therapy of solid tumors.

Ultrasound Med Biol. 2010 Mar;36(3):430-40

Authors: Casey G, Cashman JP, Morrissey D, Whelan MC, Larkin JO, Soden DM, Tangney M, O'Sullivan GC

Development of gene-based therapies for the treatment of inherited and acquired diseases, including cancer, has seen renewed interest in the use of nonviral vectors coupled to physical delivery modalities. Low-frequency ultrasound (US), with a well-established record in a clinical setting, has the potential to deliver DNA efficiently, accurately and safely. Optimal in vivo parameters for US-mediated delivery of naked plasmid DNA were established using the firefly luciferase reporter gene construct. Optimized parameters were used to administer a therapeutic gene construct, coding for granulocyte-macrophage colony-stimulating factor (GM-CSF) and B7-1 costimulatory molecule, to growing murine fibrosarcoma tumors. Tumor progression and animal survival was monitored throughout the study and the efficacy of the US-mediated gene therapy determined and compared with an electroporation-based approach. Optimal parameters for US-mediated delivery of plasmid DNA to tumors were deduced to be 1.0 W/cm(2) at 20% duty cycle for 5 min (60 J/cm(2)). In vivo US-mediated gene therapy resulted in a 55% cure rate in tumor-bearing animals. The immunological response invoked was cell mediated, conferring resistance against re-challenge and resistance to tumor challenge after transfer of splenocytes to naïve animals. US treatment was noninjurious to treated tissue, whereas therapeutic efficacy was comparable to an electroporation-based approach. US-mediated delivery of an immune-gene construct to growing tumors was therapeutically effective. Sonoporation has the potential to be a major factor in the development of nonviral gene delivery approaches.

PMID: 20133039 [PubMed - indexed for MEDLINE]