Aluminum particles generated during millisecond electric pulse application enhance adenovirus-mediated gene transfer in L929 cells.

Gene electrotransfer is an attractive method of non-viral gene delivery. However, the mechanism of DNA penetration across the plasma membrane is widely discussed. To explore this process for even larger structures, like viruses, we applied various combinations of short/long and high/low-amplitude electric pulses to L929 cells, mixed with a human adenovirus vector expressing GFP. We observed a transgene expression increase, both in the number of GFP-converted cells and GFP levels, when we added a low-voltage/millisecond-pulse treatment to the adenovirus/cell mixture. This increase, reflecting enhanced virus penetration, was proportional to the applied electric field amplitude and pulse number, but was not associated with membrane permeabilization, nor to direct cell modifications. We demonstrated that this effect is mainly due to adenovirus particle interactions with aggregated aluminum particles released from energized electrodes. Indeed, after centrifugation of the pulsed viral suspension and later on addition to cells, the activity was found mainly associated with the aluminum aggregates concentrated in the lower fraction and was proportional to generated quantities. Overall, this work focused on the use of electrotransfer to facilitate the adenovirus entry into cell, demonstrating that modifications of the penetrating agent can be more important than modifications of the target cell for transfer efficacy.

Apoptosis induction by combination of drugs or a conjugated molecule associating non-steroidal anti-inflammatory and nitric oxide donor effects in medullary thyroid cancer models: implication of the tumor suppressor p73.

BACKGROUND: Medullary thyroid cancer (MTC) is a C-cell neoplasm. Surgery remains its main treatment. Promising therapies based on tyrosine kinase inhibitors demand careful patient selection. We previously observed that two non-steroidal anti-inflammatory drugs (NSAID), indomethacin, celecoxib, and nitric oxide (NO) prevented tumor growth in a model of human MTC cell line (TT) in nude mice. METHODS: In the present study, we tested the NO donor: glyceryl trinitrate (GTN), at pharmacological dose, alone and in combination with each of the two NSAIDs on TT cells. We also assessed the anti-proliferative potential of NO-indomethacin, an indomethacin molecule chemically conjugated with a NO moiety (NCX 530, Nicox SA) on TT cells and indomethacin/GTN association in rMTC 6-23 cells. The anti-tumoral action of the combined sc. injections of GTN with oral delivery of indomethacin was also studied on subcutaneous TT tumors in nude mice. Apoptosis mechanisms were assessed by expression of caspase-3, TAp73α, TAp73α inhibition by siRNA or Annexin V externalisation. RESULTS: The two NSAIDs and GTN reduced mitotic activity in TT cells versus control (cell number and PCNA protein expression). The combined treatments amplified the anti-tumor effect of single agents in the two tested cell lines and promoted cell death. Moreover, indomethacin/GTN association stopped the growth of established TT tumors in nude mice. We observed a significant cleavage of full length PARP, a caspase-3 substrate. The cell death appearance was correlated with a two-fold increase in TAp73α expression, with inhibition of apoptosis after TAp73α siRNA addition, demonstrating its crucial role in apoptosis. CONCLUSION: Association of NO with NSAID exhibited amplified anti-tumoral effects on in vitro and in vivo MTC models by inducing p73-dependent apoptotic cell death.

Detrimental arterial inflammatory effect of microparticles circulating in preeclamptic women: ex vivo evaluation in human arteries.

Elevated plasmatic levels of lympho-monocyte and platelet microparticles (MPs) have been reported in preeclampsia. Previous studies suggest that MPs could participate in preeclampsia vascular impairment. In this study, we investigated the ex vivo vascular effects of MPs from preeclamptic women on arteries from normotensive pregnant women. Omental arteries were collected from normal pregnant women undergoing cesarean section and incubated during 24 h with MPs from normal pregnant or preeclamptic women. Vascular contraction to serotonin and phenylephrine was studied on a wire myograph with or without pharmacological selective inhibitors of inducible nitric oxide synthase (iNOS) and/or cyclo-oxygenase-2 (COX-2). Expression of iNOS, COX-2, and NF-κB and production of superoxide anion and 8-isoprostane were also assessed by immunohistological or biochemical staining and/or Western blot or ELISA assay, respectively. Microparticles from preeclamptic women, but not those from normal pregnant women, induced hyporeactivity to vasocontracturant agonists in omental arteries. Selective inhibitor of iNOS partially restored this arterial contraction, suggesting that nitric oxide (NO) is involved in vascular contractility alteration. Conversely, COX-2 induced 8-isoprostane release, a vasoconstricting metabolite modulating the agonist-induced contraction. COX-2 selective inhibitor almost abolished the arterial contraction in the same vessels. Interestingly, the association of iNOS and COX-2 selective inhibitors restored the contraction to control levels. Moreover, iNOS, COX-2, and NF-κB expressions are upregulated and superoxide anion levels increased in vessels incubated with MPs from preeclamptic women. In conclusion, circulating MPs from preeclamptic women induce vascular inflammation and enhance oxidative stress. These results suggest a possible role of MPs during preeclampsia-induced arterial dysfunction.

Cyclooxygenase-2-derived prostacyclin protective role on endotoxin-induced mouse cardiomyocyte mortality.

Cardiovascular dysfunction characterizes septic shock, inducing multiple organ failure and a high mortality rate. In the heart, it has been shown an up-regulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions with subsequent overproduction of nitric oxide (NO) and eicosanoids. This study is focused on the links between these products of inflammation and cell loss of mouse cardiomyocytes during treatment by the Salmonella typhimurium lipopolysaccharide (LPS) in presence or in absence of NOS or COX inhibitors. LPS induced RelA/NF-κB p65 activation, iNOS and COX-2 up-regulations, resulting in NO and prostacyclin releases. These effects were reversed by the NO-synthase inhibitor and increased by the specific COX-2 inhibitor. Immunostainings with FITC-conjugated anti-Annexin-V and propidium iodide and caspase 3/7 activity assay showed that cardiomyocyte necrosis was inhibited by L-NA during LPS treatment challenge, while apoptosis was induced in presence of both LPS and NS-398. No effect on LPS cellular injury was observed using the specific cyclooxygenase-1 (COX-1) inhibitor, SC-560. These findings strongly support the hypothesis of a link between iNOS-dependent NO overproduction and LPS-induced cell loss with a selective protective role allotted to COX-2 and deriving prostacyclins.

PPARδ activity in cardiovascular diseases: A potential pharmacological target.

Activation of peroxisome proliferator-activated receptors (PPARs), and particularly of PPARα and PPARγ, using selective agonists, is currently used in the treatment of metabolic diseases such as hypertriglyceridemia and type 2 diabetes mellitus. PPARα and PPARγ anti-inflammatory, antiproliferative and antiangiogenic properties in cardiovascular cells were extensively clarified in a variety of in vitro and in vivo models. In contrast, the role of PPARδ in cardiovascular system is poorly understood. Prostacyclin, the predominant prostanoid released by vascular cells, is a putative endogenous agonist for PPARδ, but only recently PPARδ selective synthetic agonists were found, improving studies about the physiological and pathophysiological roles of PPARδ activation. Recent reports suggest that the PPARδ activation may play a pivotal role to regulate inflammation, apoptosis, and cell proliferation, suggesting that this transcriptional factor could become an interesting pharmacological target to regulate cardiovascular cell apoptosis, proliferation, inflammation, and metabolism.

Percutaneous intracoronary delivery of SERCA gene increases myocardial function: a tissue Doppler imaging echocardiographic study.

The aim of this study was to examine the efficiency of adenovirus-mediated overexpression of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1a) gene in a realistic model based on percutaneous intracoronary delivery and on noninvasive functional monitoring. Catheter-based selective coronary delivery of saline or adenoviruses (Ad.CMV.SERCA1a or Ad.CMV.lacZ, 10(10) plaque-forming units) was performed in the circumflex artery of rabbits. Effects were assessed and compared by using serial Doppler echocardiography, hemodynamics, and measurements of SERCA protein and Ca(2+) uptake activity. On day 3, a 21% increase in SERCA proteins and a 37% increase in the maximal rate of Ca(2+) uptake were observed in the transfected left ventricular (LV) walls of Ad.CMV.SERCA1a rabbits. Baseline hemodynamics and conventional echographic measurements of global LV function were poorly affected. In contrast, tissue Doppler imaging (TDI) was able to assess a strong increase in the baseline function of transfected LV walls, as assessed with maximal wall velocities (+32% and +43%, respectively) and strain rates (+18% and +30%, respectively). TDI parameters were closely related to the maximal rate of Ca(2+) uptake (r(2) = 0.68 for the systolic strain rate). Serial TDI analysis during follow-up showed that the effects lasted for 7 days and were no longer detectable 15 days after adenoviruses injection. In conclusion, LV function can be increased by adenovirus-mediated overexpression of SERCA in a clinically relevant model, and TDI provides an accurate and noninvasive tool for monitoring effects on global as well as regional myocardial function.

Medullary thyroid carcinoma arises in the absence of prolactin signaling.

Prolactin, a pituitary hormone, exerts pleiotropic effects in various cells. These effects are mediated by a membrane receptor highly expressed in many tissues. To analyze prolactin effects on the thyroid gland, we first identified prolactin receptor (PRLR) mRNAs by in situ hybridization. To further evaluate the physiologic relevance of PRLR actions in the thyroid in vivo, we used PRLR knockout mice. Whereas the histologic structure of thyroid of PRLR-null mice was not disturbed, we show that T4 levels are lower in null animals (13.63 +/- 2.98 versus 10.78 +/- 2.25 pmol/L in null mice), confirming that prolactin participates in the control of thyroid metabolism. To further investigate thyroid effects in mice, we measured body temperature and thyroid-stimulating hormone in young and adult male and/or female PRLR-null mice and their normal siblings. Surprisingly, in null animals, we saw medullary thyroid carcinoma (MTC) arising from parafollicular C cells producing calcitonin. The incidence of these carcinomas attained 41% in PRLR-null mice, whereas this malignant tumor occurs sporadically or as a component of the familial cancer syndrome in humans. This finding suggests that PRLR-null mice could represent a valuable animal model for MTC, which could be compared with existing MTC models. These observations suggest a possible link between the appearance of this carcinoma and the absence of prolactin signaling.

Adenovirus-mediated gene transfer to the transplanted piglet heart after intracoronary injection.

BACKGROUND: The advent of cardiac gene therapy in clinical practice requires a more efficient and safer myocardial gene delivery in large animals. A new approach to adenovirus-mediated intracoronary gene transfer in the piglet, using a heterotopic heart transplantation model, was designed to maximize the duration of contact between the vector and the heart in noncoronary flow conditions. METHODS: Recombinant adenoviruses harboring a nucleus-localized beta-galactosidase gene under the control of a viral promoter were injected into the coronary vessels of the harvested hearts at a dose ranging from 10(10) to 2 x 10(11) pfu. The graft was maintained for 75 min in saline solution and then implanted in the abdomen of recipients. Gene transfer to allografts was evaluated 4 days after grafting by immunohistochemical and enzymatic analysis of beta-galactosidase expression. RESULTS: Transgene expression was detected in all cardiac areas and up to 64, 44, 32, and 15% of positive nuclei were estimated in the left ventricle wall in four animals out of eleven. In the remaining animals, transgene expression was focally distributed, mainly in the left ventricle wall. PCR analysis revealed the presence of adenoviral sequences, albeit minimal, in exposed organs such as the liver and lung. CONCLUSIONS: This procedure demonstrated that direct intracoronary gene transfer can be achieved using an ex vivo gene transfer strategy.