FAK alternative splice mRNA variants expression pattern in colorectal cancer.

The Focal adhesion kinase (FAK) is a ubiquitous cytoplasmic tyrosine-kinase promoting tumor progression and metastasis processes by acting in cancer cells and their tumor microenvironment partners. FAK overexpression in primary colon tumors and their metastasis is associated to poor colorectal cancer (CRC) patients' outcome. Eight FAK mRNA alternative splice variants have been described and contribute to additional level of FAK activity regulation, some of them corresponding to overactivated FAK isoforms. To date, FAK mRNA alternative splice variants expression and implication in CRC processes remain unknown. Here, using different human CRC cells lines displaying differential invasive capacities in an in vivo murine model recapitulating the different steps of CRC development from primary tumors to liver and lung metastasis, we identified three out of the eight mRNA variants (namely FAK0 , FAK28 and FAK6 ) differentially expressed along the CRC process and the tumor sites. Our results highlight an association between FAK0 and FAK6 expressions and the metastatic potential of the most aggressive cell lines HT29 and HCT116, suggesting that FAK0 and FAK6 could represent aggressiveness markers in CRC. Our findings also suggest a more specific role for FAK28 in the interactions between the tumors cells and their microenvironment. In conclusion, targeting FAK0 , the common form of FAK, might not be a good strategy based on the numerous roles of this kinase in physiological processes. In contrast, FAK6 or FAK28 splice variants, or their corresponding protein isoforms, may putatively represent future therapeutic target candidates in the development of CRC primary tumors and metastasis.

Mitochondrial HSP90 Accumulation Promotes Vascular Remodeling in Pulmonary Arterial Hypertension.

RATIONALE: Pulmonary arterial hypertension (PAH) is a vascular remodeling disease with a poor prognosis and limited therapeutic options. Although the mechanisms contributing to vascular remodeling in PAH are still unclear, several features, including hyperproliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs), have led to the emergence of the cancer-like concept. The molecular chaperone HSP90 (heat shock protein 90) is directly associated with malignant growth and proliferation under stress conditions. In addition to being highly expressed in the cytosol, HSP90 exists in a subcellular pool compartmentalized in the mitochondria (mtHSP90) of tumor cells, but not in normal cells, where it promotes cell survival. OBJECTIVES: We hypothesized that mtHSP90 in PAH-PASMCs represents a protective mechanism against stress, promoting their proliferation and resistance to apoptosis. METHODS: Expression and localization of HSP90 were analyzed by Western blot, immunofluorescence, and immunogold electron microscopy. In vitro, effects of mtHSP90 inhibition on mitochondrial DNA integrity, bioenergetics, cell proliferation and resistance to apoptosis were assessed. In vivo, the therapeutic potential of Gamitrinib, a mitochondria-targeted HSP90 inhibitor, was tested in fawn-hooded and monocrotaline rats. MEASUREMENTS AND MAIN RESULTS: We demonstrated that, in response to stress, HSP90 preferentially accumulates in PAH-PASMC mitochondria (dual immunostaining, immunoblot, and immunogold electron microscopy) to ensure cell survival by preserving mitochondrial DNA integrity and bioenergetic functions. Whereas cytosolic HSP90 inhibition displays a lack of absolute specificity for PAH-PASMCs, Gamitrinib decreased mitochondrial DNA content and repair capacity and bioenergetic functions, thus repressing PAH-PASMC proliferation (Ki67 labeling) and resistance to apoptosis (Annexin V assay) without affecting control cells. In vivo, Gamitrinib improves PAH in two experimental rat models (monocrotaline and fawn-hooded rat). CONCLUSIONS: Our data show for the first time that accumulation of mtHSP90 is a feature of PAH-PASMCs and a key regulator of mitochondrial homeostasis contributing to vascular remodeling in PAH.

Bromodomain-Containing Protein 4: The Epigenetic Origin of Pulmonary Arterial Hypertension.

RATIONALE: Pulmonary arterial hypertension (PAH) is a vasculopathy characterized by enhanced pulmonary artery (PA) smooth muscle cell (PASMC) proliferation and suppressed apoptosis. Decreased expression of microRNA-204 has been associated to this phenotype. By a still elusive mechanism, microRNA-204 downregulation promotes the expression of oncogenes, including nuclear factor of activated T cells, B-cell lymphoma 2, and Survivin. In cancer, increased expression of the epigenetic reader bromodomain-containing protein 4 (BRD4) sustains cell survival and proliferation. Interestingly, BRD4 is a predicted target of microRNA-204 and has binding sites on the nuclear factor of activated T cells promoter region. OBJECTIVE: To investigate the role of BRD4 in PAH pathogenesis. METHODS AND RESULTS: BRD4 is upregulated in lungs, distal PAs, and PASMCs of patients with PAH compared with controls. With mechanistic in vitro experiments, we demonstrated that BRD4 expression in PAH is microRNA-204 dependent. We further studied the molecular downstream targets of BRD4 by inhibiting its activity in PAH-PASMCs using a clinically available inhibitor JQ1. JQ1 treatment in PAH-PASMCs increased p21 expression, thus triggering cell cycle arrest. Furthermore, BRD4 inhibition, by JQ1 or siBRD4, decreased the expression of 3 major oncogenes, which are overexpressed in PAH: nuclear factor of activated T cells, B-cell lymphoma 2, and Survivin. Blocking this oncogenic signature led to decreased PAH-PASMC proliferation and increased apoptosis in a BRD4-dependent manner. Indeed, pharmacological JQ1 or molecular (siRNA) inhibition of BRD4 reversed this pathological phenotype in addition to restoring mitochondrial membrane potential and to increasing cells spare respiratory capacity. Moreover, BRD4 inhibition in vivo reversed established PAH in the Sugen/hypoxia rat model. CONCLUSIONS: BRD4 plays a key role in the pathological phenotype in PAH, which could offer new therapeutic perspectives for patients with PAH.

Role for Runt-related Transcription Factor 2 in Proliferative and Calcified Vascular Lesions in Pulmonary Arterial Hypertension.

RATIONALE: Pulmonary arterial hypertension (PAH) is characterized by excessive proliferation of pulmonary artery smooth muscle cells (PASMCs). This is sustained in time by the down-regulation of microRNA (miR)-204. In systemic vascular diseases, reduced miR-204 expression promotes vascular biomineralization by augmenting the expression of the transcription factor Runt-related transcription factor 2 (RUNX2). Implication of RUNX2 in PAH-related vascular remodeling and presence of calcified lesions in PAH remain unexplored. OBJECTIVES: We hypothesized that RUNX2 is up-regulated in lungs of patients with PAH, contributing to vascular remodeling and calcium-related biomineralization. METHODS: We harvested human lung tissues in which we assessed calcification lesions and RUNX2 expression. We also isolated PASMCs from these tissues for in vitro analyses. Using a bidirectional approach, we investigated the role for RUNX2 in cell proliferation, apoptosis, and calcification capacity. Ectopic delivery of small interfering RNA against RUNX2 was used in an animal model of PAH to evaluate the therapeutic potential of RUNX2 inhibition in this disease. MEASUREMENTS AND MAIN RESULTS: Patients with PAH display features of calcified lesions within the distal pulmonary arteries (PAs). We show that RUNX2 is up-regulated in lungs, distal PAs, and primary cultured human PASMCs isolated from PAH and compared with patients without PAH. RUNX2 expression histologically correlates with vascular remodeling and calcification. Using in vitro gain- and loss-of-function approaches, we mechanistically demonstrate that miR-204 diminution promotes RUNX2 up-regulation and that sustained RUNX2 expression activates hypoxia-inducible factor-1α, leading to aberrant proliferation, resistance to apoptosis, and subsequent transdifferentiation of PAH-PASMCs into osteoblast-like cells. In the PAH Sugen/hypoxia rat model, molecular RUNX2 inhibition reduces PA remodeling and prevents calcification, thus improving pulmonary hemodynamic parameters and right ventricular function. CONCLUSIONS: RUNX2 plays a pivotal role in the pathogenesis of PAH, contributing to the development of proliferative and calcified PA lesions. Inhibition of RUNX2 may therefore represent an attractive therapeutic strategy for PAH.

Potassium channels in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a devastating cardiopulmonary disorder with various origins. All forms of PAH share a common pulmonary arteriopathy characterised by vasoconstriction, remodelling of the pre-capillary pulmonary vessel wall, and in situ thrombosis. Although the pathogenesis of PAH is recognised as a complex and multifactorial process, there is growing evidence that potassium channels dysfunction in pulmonary artery smooth muscle cells is a hallmark of PAH. Besides regulating many physiological functions, reduced potassium channels expression and/or activity have significant effects on PAH establishment and progression. This review describes the molecular mechanisms and physiological consequences of potassium channel modulation. Special emphasis is placed on KCNA5 (Kv1.5) and KCNK3 (TASK1), which are considered to play a central role in determining pulmonary vascular tone and may represent attractive therapeutic targets in the treatment of PAH.

LNA-based oligonucleotide electrotransfer for miRNA inhibition.

Micro-RNAs (miRNAs) are small regulatory RNAs that play an important role in disease development and progression and therefore represent a potential new class of therapeutic targets. However, an effective and safe clinical approach for miRNA inhibition remains elusive, primarily due to the lack of effective delivery methods. We proposed to inhibit miRNA by electrotransferring an antisense DNA oligomer containing locked nucleic acids (LNAs) (LNA/DNA oligomer). We observed that electropulsation (EP) led to a strong cellular uptake of LNA/DNA oligomer. The LNA/DNA oligomer electrotransfer mechanism and intracellular localization were visually investigated in real time at the single-cell level. Cyanine 5-labeled oligonucleotide entered exclusively during pulse application on the side of the permeabilized cell membrane facing the cathode, driven by electrophoretic forces. Minutes after the electrotransfer, the LNA/DNA oligomer diffused into the nucleus. EP provided the anti-miRNA oligomer with immediate and direct access to its cytoplasmic mature miRNA target and/or its nuclear precursor miRNA target. We then demonstrated using a LNA/DNA oligomer anti-miR34a that LNA/DNA oligomer electrotransfer decreased the level of the miR34a target and induced its functional inhibition. Our findings show that using the electrotransfer technique for LNA-based oligonucleotide delivery is a promising therapeutic strategy to silence deleterious miRNAs overexpressed in diseases.

Chemically modified oligonucleotide-increased stability negatively correlates with its efficacy despite efficient electrotransfer.

Despite great potential for disease treatment, small interfering RNA (siRNA) development has been hampered due to its poor stability and the lack of efficient delivery method. To overcome the sensitivity, new generations of chemically modified oligonucleotides have been developed such as the locked nucleic acid (LNA). LNA substitution in an siRNA sequence (siLNA) is supposed to increase its stability and its affinity for its complementary sequence. The purpose of this study was to evaluate the potential benefit of an anti-GFP siLNA using the biophysical delivery method electropermeabilization. We used two types of electrical conditions: electrochemotherapy (ECT), a condition for efficient transfer of small molecules in clinics, and electrogenotherapy (EGT), a condition for efficient transfer of macromolecules. We first confirmed that siLNA was indeed more stable in mouse serum than unmodified siRNA. After determining the ECT and EGT optimal electrical parameters for a human colorectal carcinoma cell line (HCT-116) expressing eGFP, we showed that modifications of siRNA do not interfere with electrotransfer efficiency. However, despite its higher stability and its high electrotransfer efficacy, siLNA was less efficient for eGFP silencing compared to the electrotransferred, unmodified siRNA regardless of the electrical conditions used. Our study highlighted the care that is needed when designing chemically modified oligonucleotides.

Pain Management in a Model of Interstitial Cystitis/Bladder Pain Syndrome by a Vaccinal Strategy.

Current analgesic treatments for Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS) are limited. Here, we propose a novel antinociceptive strategy exploiting the opioid-mediated analgesic properties of T lymphocytes to relieve from bladder pain. In a chronic model of IC/BPS in rats, we show that a secondary T cell response against intravesically administered ovalbumin prevents from visceral pain in OVA-primed animals. The analgesic effect is associated with the recruitment of T lymphocytes within the inflamed mucosa and is reversed by naloxone-methiodide, a peripheral opioid receptor antagonist. Similarly, intravesical instillation of BCG or tetanus toxoid antigens in vaccinated rats protects from pain in the same model. We show opioid-dependent analgesic properties of local vaccine antigen recall in a preclinical rat model of chronic cystitis. Since BCG bladder instillation is regularly used in humans (as anticancer therapy), our results open it as a new therapeutic positioning for a pain management indication for IC/BPS patients.

Characterization and Validation of a Chronic Model of Cyclophosphamide-Induced Interstitial Cystitis/Bladder Pain Syndrome in Rats.

Interstitial cystitis/Bladder Pain Syndrome (IC/BPS) is a chronic inflammatory disease characterized by visceral pain and voiding symptoms. IC/BPS is still an unsolved enigma with ineffective diagnosis criteria and treatment. A main limitation in IC/BPS understanding is the lack of appropriate preclinical model. Cyclophosphamide (CYP) is commonly used as an experimental model for IC/BPS in rodent. However, the proposed models are very aggressive, contrasting with what occurs in clinic, and often associated with severe toxicity and high mortality rate. In addition, visceral pain, the hallmark symptom of IC/BPS, has been validated in only few of them. In this study, we developed a chronic model of CYP-induced IC/BPS in female rat. In our protocol, no severe weight loss occurred and the survival rate was 100%. In accordance to human pathology, chronic CYP-injected rats developed severe painful behavior whereas only sparse inflammation was observed. Inflammatory response was characterized by bladder edema and focal urothelial damage but absence of massive infiltrate. This chronic model showed persistent symptoms indicative for a central sensitization mechanism. We further demonstrate that CYP-induced chronic visceral pain was significantly reduced by curative treatment with clinically relevant compounds (gabapentin, ibuprofen, and Ialuril®). We therefore developed and validated a rat model of chronic cystitis that shares strong similarity with human non-ulcerative IC/BPS features without overtly affecting the animal health. This model will thus provide mechanistic insights of the disease and help to evaluate therapeutic agents for IC/BPS.

Development of a near infrared protein nanoprobe targeting Thomsen-Friedenreich antigen for intraoperative detection of submillimeter nodules in an ovarian peritoneal carcinomatosis mouse model.

The epithelial ovarian cancer is one of the most lethal gynecological malignancy due to its late diagnostic and many relapses observed after first line of treatment. Once diagnose, the most important prognostic factor is the completeness of cytoreductive surgery. To achieve this goal, surgeons have to pinpoint and remove nodules, especially the smallest nodules. Recent advances in fluorescence-guided surgery led us to develop a recombinant lectin as a nanoprobe for the microscopic detection of nodules in the peritoneal cavity of tumor-bearing mice. This lectin has an intrinsic specificity for a carcinoma-associated glycan biomarker, the Thomsen-Friedenreich antigen. In this study, after its labelling by a near infrared dye, we first demonstrated that this nanoprobe allowed indirect detection of nodules already implanted in the peritoneal cavity, through tumor microenvironment targeting. Secondly, in a protocol mimicking the scattering of cells during surgery, we obtained a direct and long-lasting detection of tumor cells in vivo. This lectin as already been described as a nanocontainer able to do targeted delivery of a therapeutic compound to carcinoma cells. Future developments will focus on the combination of the nanoprobe and nanocontainer aspects in an intraperitoneal nanotheranostic approach.

Characterization of Human Colon Organoids From Inflammatory Bowel Disease Patients.

Inflammatory Bowel Diseases (IBD) are chronic inflammatory disorders, where epithelial defects drive, at least in part, some of the pathology. We reconstituted human intestinal epithelial organ, by using three-dimension culture of human colon organoids. Our aim was to characterize morphological and functional phenotypes of control (non-IBD) organoids, compared to inflamed organoids from IBD patients. The results generated describe the epithelial defects associated with IBD in primary organoid cultures, and evaluate the use of this model for pharmacological testing of anti-inflammatory approaches. Human colonic tissues were obtained from either surgical resections or biopsies, all harvested in non-inflammatory zones. Crypts were isolated from controls (non-IBD) and IBD patients and were cultured up to 12-days. Morphological (size, budding formation, polarization, luminal content), cell composition (proliferation, differentiation, immaturity markers expression), and functional (chemokine and tight junction protein expression) parameters were measured by immunohistochemistry, RT-qPCR or western-blot. The effects of inflammatory cocktail or anti-inflammatory treatments were studied in controls and IBD organoid cultures respectively. Organoid cultures from controls or IBD patients had the same cell composition after 10 to 12-days of culture, but IBD organoid cultures showed an inflammatory phenotype with decreased size and budding capacity, increased cell death, luminal debris, and inverted polarization. Tight junction proteins were also significantly decreased in IBD organoid cultures. Inflammatory cytokine cocktail reproduced this inflammatory phenotype in non-IBD organoids. Clinically used treatments (5-ASA, glucocorticoids, anti-TNF) reduced some, but not all parameters. Inflammatory phenotype is associated with IBD epithelium, and can be studied in organoid cultures. This model constitutes a reliable human pre-clinical model to investigate new strategies targeting epithelial repair.

Noninvasive Gene Electrotransfer in Skin.

The skin is considered as well suited for gene therapy and vaccination. DNA vaccines elicit both broad humoral and cellular immune responses when injected in the skin. Physical and chemical methods are needed to boost the expression. Gene electrotransfer (GET) is one of the most effective approaches. This step-by-step protocol describes the procedures to obtain an efficient GET targeted to the skin by using easy-to-use noninvasive electrodes after intradermal plasmid injection (i.d. GET). A specific pulse sequence is reported. Expression is observed by in vivo fluorescence imaging during >2 weeks as the plasmid was coding for tdTomato. The protocol is efficient for the transient expression of clinical proteins.

Pre-clinical investigation of the synergy effect of interleukin-12 gene-electro-transfer during partially irreversible electropermeabilization against melanoma.

BACKGROUND: Melanoma is a very aggressive skin tumor that can be cured when diagnosed and treated in its early stages. However, at the time of identification, the tumor is frequently in a metastatic stage. Intensive research is currently ongoing to improve the efficacy of the immune system in eliminating cancer cells. One approach is to boost the activation of cytotoxic T cells by IL-12 cytokine that plays a central role in the activation of the immune system. In parallel, physical methods such as electropermeabilization-based treatments are currently under investigation and show promising results. METHODS: In this study, we set electrical parameters to induce a partial-irreversible electropermeabilization (pIRE) of melanoma to induce a sufficient cell death and potential release of tumor antigens able to activate immune cells. This protocol mimics the situation where irreversible electropermeabilization is not fully completed. Then, a peritumoral plasmid IL-12 electrotransfer was combined with pIRE treatment. Evaluation of the tumor growth and survival was performed in mouse strains having a different immunological background (C57Bl/6 (WT), nude and C57Bl6 (TLR9-/-)). RESULTS: pIRE treatment induced apoptotic cell death and a temporary tumor growth delay in all mouse strains. In C57Bl/6 mice, we showed that peritumoral plasmid IL-12 electrotransfer combined with tumor pIRE treatment induced tumor regression correlating with a local secretion of IL-12 and IFN-γ. This combined treatment induced a growth delay of distant tumors and prevented the emergence of a second tumor in 50% of immunocompetent mice. CONCLUSIONS: The combination of pIL-12 GET and pIRE not only enhanced survival but could bring a curative effect in wild type mice. This two-step treatment, named Immune-Gene Electro-Therapy (IGET), led to a systemic activation of the adaptive immune system and the development of an anti-tumor immune memory.

Retinoids inhibit measles virus in vitro via nuclear retinoid receptor signaling pathways.

Measles virus (MV) infects 30 million children every year, resulting in more than half a million deaths. Vitamin A (retinol) treatment of acute measles can reduce measles-associated mortality by 50-80%. We sought to determine whether or not retinoids can act directly to limit MV output from infected cells. Physiologic concentrations of retinol were found to inhibit MV output in PBMC and a range of cell lines of epithelial and endothelial origin (40-50%). Near complete inhibition of viral output was achieved in some cells/lines treated with all-trans retinoic acid (ATRA) and 9-cis RA (9cRA). Important attenuation of the anti-MV effect of retinoids in R4 cells, a subclone of a retinoid-responsive cell line (NB4) deficient in RAR signaling, demonstrates that this effect is mediated at least in part by nuclear retinoid receptor signaling pathways. Inhibition of MV replication could not be fully explained as a result of retinoid effects on cell differentiation, proliferation or viability, particularly at low retinoid concentrations (1-10nM). These data provide the first evidence that retinoids can directly inhibit MV in vitro, and raise the possibility that retinoids may have similar actions in vivo.

Safe and efficient novel approach for non-invasive gene electrotransfer to skin.

Gene transfer into cells or tissue by application of electric pulses (i.e. gene electrotransfer (GET)) is a non-viral gene delivery method that is becoming increasingly attractive for clinical applications. In order to make GET progress to wide clinical usage its efficacy needs to be improved and the safety of the method has to be confirmed. Therefore, the aim of our study was to increase GET efficacy in skin, by optimizing electric pulse parameters and the design of electrodes. We evaluated the safety of our novel approach by assaying the thermal stress effect of GET conditions and the biodistribution of a cytokine expressing plasmid. Transfection efficacy of different pulse parameters was determined using two reporter genes encoding for the green fluorescent protein (GFP) and the tdTomato fluorescent protein, respectively. GET was performed using non-invasive contact electrodes immediately after intradermal injection of plasmid DNA into mouse skin. Fluorescence imaging of transfected skin showed that a sophistication in the pulse parameters could be selected to get greater transfection efficacy in comparison to the standard ones. Delivery of electric pulses only mildly induced expression of the heat shock protein Hsp70 in a luminescent reporting transgenic mouse model, demonstrating that there were no drastic stress effects. The plasmid was not detected in other organs and was found only at the site of treatment for a limited period of time. In conclusion, we set up a novel approach for GET combining new electric field parameters with high voltage short pulses and medium voltage long pulses using contact electrodes, to obtain a high expression of both fluorescent reporter and therapeutic genes while showing full safety in living animals.

Thrombin modifies growth, proliferation and apoptosis of human colon organoids: a protease-activated receptor 1- and protease-activated receptor 4-dependent mechanism.

BACKGROUND AND PURPOSE: Thrombin is massively released upon tissue damage associated with bleeding or chronic inflammation. The effects of this thrombin on tissue regrowth and repair has been scarcely addressed and only in cancer cell lines. Hence, the purpose of the present study was to determine thrombin's pharmacological effects on human intestinal epithelium growth, proliferation and apoptosis, using three-dimensional cultures of human colon organoids. EXPERIMENTAL APPROACH: Crypts were isolated from human colonic resections and cultured for 6 days, forming human colon organoids. Cultured organoids were exposed to 10 and 50 mU·mL-1 of thrombin, in the presence or not of protease-activated receptor (PAR) antagonists. Organoid morphology, metabolism, proliferation and apoptosis were followed. KEY RESULTS: Thrombin favoured organoid maturation leading to a decreased number of immature cystic structures and a concomitant increased number of larger structures releasing cell debris and apoptotic cells. The size of budding structures, metabolic activity and proliferation were significantly reduced in organoid cultures exposed to thrombin, while apoptosis was dramatically increased. Both PAR1 and PAR4 antagonists inhibited apoptosis regardless of thrombin doses. Thrombin-induced inhibition of proliferation and metabolic activity were reversed by PAR4 antagonist for thrombin's lowest dose and by PAR1 antagonist for thrombin's highest dose. CONCLUSIONS AND IMPLICATIONS: Overall, our data suggest that the presence of thrombin in the vicinity of human colon epithelial cells favours their maturation at the expense of their regenerative capacities. Our data point to thrombin and its two receptors PAR1 and PAR4 as potential molecular targets for epithelial repair therapies.

Soluble HLA-G and control of angiogenesis.

In this review, we summarize the results of a number of our recent in vitro and in vivo experiments demonstrating that, in addition to the immunoregulatory functions, soluble HLA-G molecules also affect endothelial cell activity. We have found that soluble HLA-G1 (also designated HLA-G5) inhibits endothelial cell proliferation, migration and tubule formation, and this occurred through binding to the CD160 receptor and via an apoptotic pathway. Moreover, we have demonstrated that soluble HLA-G1 blocks in vivo rabbit corneal neoangiogenesis. Although it cannot be excluded that other soluble HLA class I molecules may have similar effects, as soluble forms of HLA-G are being produced by trophoblast cells at the maternal-fetal interface during early gestation, we discuss how such anti-angiogenic properties of soluble HLA-G1 may locally influence uterine vascular remodeling.

Traditional dietary additives of the Maasai are antiviral against the measles virus.

Although ethnopharmacological investigations emphasize the importance of medicinal plants in developing countries, species used regularly with diet are under-investigated and potentially make greater contributions to health. Thirteen traditional plants most commonly added to milk/soups by the Maasai for perceived health benefits were tested for activity against measles virus (MV) using non-medicinal plants as controls. Antiviral effects of plant extracts were sought using a modified neutralization assay. Methanolic extracts of medicinal species exhibited significantly greater activity neutralizing MV in vitro in comparison to non-medicinal extracts (p<0.02). Four of 13 (31%) medicinal species versus 0/13 controls had measurable effects against MV in vitro. Olinia rochetiana (Olkirenyi) and Warburgia ugandensis (Osokonoi) extracts were most potent with the number of plaque forming units reduced 37- and 34-fold, respectively. Given the importance of monocytes in the dissemination of MV, we assessed the capacity of a subset of plant extracts to inhibit MV growth in monocytoid cell line, U937. MV output from U937 cells was significantly reduced by four of seven medicinal plant extracts (mean reduction 48 h: 39.0+/-26.0%, range 3.5-87%; 72 h: 56.4+/-29.5%, range 14.1-103.1%) (p<0.05). This study provides evidence that medicinal plants added to the Maasai diet may contribute to the modulation of viral infections.

HDAC6: A Novel Histone Deacetylase Implicated in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a vascular remodeling disease with limited therapeutic options. Although exposed to stressful conditions, pulmonary artery (PA) smooth muscle cells (PASMCs) exhibit a "cancer-like" pro-proliferative and anti-apoptotic phenotype. HDAC6 is a cytoplasmic histone deacetylase regulating multiple pro-survival mechanisms and overexpressed in response to stress in cancer cells. Due to the similarities between cancer and PAH, we hypothesized that HDAC6 expression is increased in PAH-PASMCs to face stress allowing them to survive and proliferate, thus contributing to vascular remodeling in PAH. We found that HDAC6 is significantly up-regulated in lungs, distal PAs, and isolated PASMCs from PAH patients and animal models. Inhibition of HDAC6 reduced PAH-PASMC proliferation and resistance to apoptosis in vitro sparing control cells. Mechanistically, we demonstrated that HDAC6 maintains Ku70 in a hypoacetylated state, blocking the translocation of Bax to mitochondria and preventing apoptosis. In vivo, pharmacological inhibition of HDAC6 improved established PAH in two experimental models and can be safely given in combination with currently approved PAH therapies. Moreover, Hdac6 deficient mice were partially protected against chronic hypoxia-induced pulmonary hypertension. Our study shows for the first time that HDAC6 is implicated in PAH development and represents a new promising target to improve PAH.

Targeted electro-delivery of oligonucleotides for RNA interference: siRNA and antimiR.

For more than a decade, the understanding of RNA interference (RNAi) has been a growing field of interest. Micro-RNAs (miRNAs) are small regulatory RNAs that play an important role in disease development and progression and therefore represent a potential new class of therapeutic targets. However, delivery of RNAi-based oligonucleotides is one of the most challenging hurdles to RNAi-based drug development. Electropermeabilization (EP) is recognized as a successful non-viral method to transfer nucleic acids into living cells both in vitro and in vivo. EP is the direct application of electric pulses to cells or tissues that transiently permeabilize plasma membranes, allowing the efficient delivery of exogenous molecules. The present review focused on the mechanism of RNAi-based oligonucleotides electrotransfer, from cellular uptake to intracellular distribution. Biophysical theories on oligonucleotide electrotransfer will be also presented. The advantages and few drawbacks of EP-mediated delivery will also be discussed.