Preventing occludin tight-junction disruption via inhibition of microRNA-193b-5p attenuates viral load and influenza-induced lung injury.

Virus-induced lung injury is associated with loss of pulmonary epithelial-endothelial tight junction integrity. While the alveolar-capillary membrane may be an indirect target of injury, viruses may interact directly and/or indirectly with miRs to augment their replication potential and evade the host antiviral defense system. Here, we expose how the influenza virus (H1N1) capitalizes on host-derived interferon-induced, microRNA (miR)-193b-5p to target occludin and compromise antiviral defenses. Lung biopsies from patients infected with H1N1 revealed increased miR-193b-5p levels, marked reduction in occludin protein, and disruption of the alveolar-capillary barrier. In C57BL/6 mice, the expression of miR-193b-5p increased, and occludin decreased, 5-6 days post-infection with influenza (PR8). Inhibition of miR-193b-5p in primary human bronchial, pulmonary microvascular, and nasal epithelial cells enhanced antiviral responses. miR-193b-deficient mice were resistant to PR8. Knockdown of occludin, both in vitro and in vivo, and overexpression of miR-193b-5p reconstituted susceptibility to viral infection. miR-193b-5p inhibitor mitigated loss of occludin, improved viral clearance, reduced lung edema, and augmented survival in infected mice. Our results elucidate how the innate immune system may be exploited by the influenza virus and how strategies that prevent loss of occludin and preserve tight junction function may limit susceptibility to virus-induced lung injury.

Mesenchymal stromal (stem) cell therapy modulates miR-193b-5p expression to attenuate sepsis-induced acute lung injury.

Although mesenchymal stromal (stem) cell (MSC) administration attenuates sepsis-induced lung injury in pre-clinical models, the mechanism(s) of action and host immune system contributions to its therapeutic effects remain elusive. We show that treatment with MSCs decreased expression of host-derived microRNA (miR)-193b-5p and increased expression of its target gene, the tight junctional protein occludin (Ocln), in lungs from septic mice. Mutating the Ocln 3' untranslated region miR-193b-5p binding sequence impaired binding to Ocln mRNA. Inhibition of miR-193b-5p in human primary pulmonary microvascular endothelial cells prevents tumour necrosis factor (TNF)-induced decrease in Ocln gene and protein expression and loss of barrier function. MSC-conditioned media mitigated TNF-induced miR-193b-5p upregulation and Ocln downregulation in vitro When administered in vivo, MSC-conditioned media recapitulated the effects of MSC administration on pulmonary miR-193b-5p and Ocln expression. MiR-193b-deficient mice were resistant to pulmonary inflammation and injury induced by lipopolysaccharide (LPS) instillation. Silencing of Ocln in miR-193b-deficient mice partially recovered the susceptibility to LPS-induced lung injury. In vivo inhibition of miR-193b-5p protected mice from endotoxin-induced lung injury. Finally, the clinical significance of these results was supported by the finding of increased miR-193b-5p expression levels in lung autopsy samples from acute respiratory distress syndrome patients who died with diffuse alveolar damage.

Immunophenotypic characterization and therapeutics effects of human bone marrow- and umbilical cord-derived mesenchymal stromal cells in an experimental model of sepsis.

Sepsis is a complicated multi-system disorder characterized by a dysregulated host response to infection. Despite substantial progress in the understanding of mechanisms of sepsis, translation of these advances into clinically effective therapies remains challenging. Mesenchymal Stromal Cells (MSCs) possess immunomodulatory properties that have shown therapeutic promise in preclinical models of sepsis. The therapeutic effects of MSCs may vary depending on the source and type of these cells. In this comparative study, the gene expression pattern and surface markers of bone marrow-derived MSCs (BM-MSCs) and umbilical cord-derived MSCs (UC-MSCs) as well as their therapeutic effects in a clinically relevant mouse model of polymicrobial sepsis, cecal ligation and puncture (CLP), were investigated. The results showed remarkable differences in gene expression profile, surface markers and therapeutic potency in terms of enhancing survival and pro/anti-inflammatory responses between the two MSC types. BM-MSCs improved survival concomitant with an enhanced systemic bacterial clearance and improved inflammatory profile post CLP surgery. Despite some improvement in the inflammatory profile of the septic animals, treatment with UC-MSCs did not enhance survival or bacterial clearance. Overall, the beneficial therapeutic effects of BM-MSCs over UC-MSCs may likely be attributed to their pro-inflammatory function, and to some extent anti-inflammatory features, reflected in their gene expression pattern enhancing macrophage polarization to M1/M2 phenotypes resulting in a balanced pro- and anti-inflammatory response against polymicrobial sepsis.

Mesenchymal Stem/Stromal Cells Increase Cardiac miR-187-3p Expression in a Polymicrobial Animal Model of Sepsis.

ABSTRACT: Sepsis-induced myocardial dysfunction (MD) is an important pathophysiological feature of multiorgan failure caused by a dysregulated host response to infection. Patients with MD continue to be managed in intensive care units with limited understanding of the molecular mechanisms controlling disease pathogenesis. Emerging evidences support the use of mesenchymal stem/stromal cell (MSC) therapy for treating critically ill septic patients. Combining this with the known role that microRNAs (miRNAs) play in reversing sepsis-induced myocardial-dysfunction, this study sought to investigate how MSC administration alters miRNA expression in the heart. Mice were randomized to experimental polymicrobial sepsis induced by cecal ligation and puncture (CLP) or sham surgery, treated with either MSCs (2.5 × 105) or placebo (saline). Twenty-eight hours post-intervention, RNA was collected from whole hearts for transcriptomic and microRNA profiling. The top microRNAs differentially regulated in hearts by CLP and MSC administration were used to generate a putative mRNA-miRNA interaction network. Key genes, termed hub genes, within the network were then identified and further validated in vivo. Network analysis and RT-qPCR revealed that septic hearts treated with MSCs resulted in upregulation of five miRNAs, including miR-187, and decrease in three top hit putative hub genes (Itpkc, Lrrc59, and Tbl1xr1). Functionally, MSC administration decreased inflammatory and apoptotic pathways, while increasing cardiac-specific structural and functional, gene expression. Taken together, our data suggest that MSC administration regulates host-derived miRNAs production to protect cardiomyocytes from sepsis-induced MD.

Genetically Modified Mesenchymal Stromal/Stem Cells: Application in Critical Illness.

Critical illnesses including sepsis, acute respiratory distress syndromes, ischemic cardiovascular disorders and acute organ injuries are associated with high mortality, morbidity as well as significant health care system expenses. While these diverse conditions require different specific therapeutic approaches, mesenchymal stem/stromal cell (MSCs) are multipotent cells capable of self-renewal, tri-lineage differentiation with a broad range regenerative and immunomodulatory activities, making them attractive for the treatment of critical illness. The therapeutic effects of MSCs have been extensively investigated in several pre-clinical models of critical illness as well as in phase I and II clinical cell therapy trials with mixed results. Whilst these studies have demonstrated the therapeutic potential for MSC therapy in critical illness, optimization for clinical use is an ongoing challenge. MSCs can be readily genetically modified by application of different techniques and tools leading to overexpress or inhibit genes related to their immunomodulatory or regenerative functions. Here we will review recent approaches designed to enhance the therapeutic potential of MSCs with an emphasis on the technology used to generate genetically modified cells, target genes, target diseases and the implication of genetically modified MSCs in cell therapy for critical illness.

Extracellular Vesicles from Interferon-γ-primed Human Umbilical Cord Mesenchymal Stromal Cells Reduce Escherichia coli-induced Acute Lung Injury in Rats.

BACKGROUND: Human umbilical cord mesenchymal stromal cells possess considerable therapeutic promise for acute respiratory distress syndrome. Umbilical cord mesenchymal stromal cells may exert therapeutic effects via extracellular vesicles, while priming umbilical cord mesenchymal stromal cells may further enhance their effect. The authors investigated whether interferon-γ-primed umbilical cord mesenchymal stromal cells would generate mesenchymal stromal cell-derived extracellular vesicles with enhanced effects in Escherichia coli (E. coli) pneumonia. METHODS: In a university laboratory, anesthetized adult male Sprague-Dawley rats (n = 8 to 18 per group) underwent intrapulmonary E. coli instillation (5 × 10 colony forming units per kilogram), and were randomized to receive (a) primed mesenchymal stromal cell-derived extracellular vesicles, (b) naïve mesenchymal stromal cell-derived extracellular vesicles (both 100 million mesenchymal stromal cell-derived extracellular vesicles per kilogram), or (c) vehicle. Injury severity and bacterial load were assessed at 48 h. In vitro studies assessed the potential for primed and naïve mesenchymal stromal cell-derived extracellular vesicles to enhance macrophage bacterial phagocytosis and killing. RESULTS: Survival increased with primed (10 of 11 [91%]) and naïve (8 of 8 [100%]) mesenchymal stromal cell-derived extracellular vesicles compared with vehicle (12 of 18 [66.7%], P = 0.038). Primed-but not naïve-mesenchymal stromal cell-derived extracellular vesicles reduced alveolar-arterial oxygen gradient (422 ± 104, 536 ± 58, 523 ± 68 mm Hg, respectively; P = 0.008), reduced alveolar protein leak (0.7 ± 0.3, 1.4 ± 0.4, 1.5 ± 0.7 mg/ml, respectively; P = 0.003), increased lung mononuclear phagocytes (23.2 ± 6.3, 21.7 ± 5, 16.7 ± 5 respectively; P = 0.025), and reduced alveolar tumor necrosis factor alpha concentrations (29 ± 14.5, 35 ± 12.3, 47.2 ± 6.3 pg/ml, respectively; P = 0.026) compared with vehicle. Primed-but not naïve-mesenchymal stromal cell-derived extracellular vesicles enhanced endothelial nitric oxide synthase production in the injured lung (endothelial nitric oxide synthase/ß-actin = 0.77 ± 0.34, 0.25 ± 0.29, 0.21 ± 0.33, respectively; P = 0.005). Both primed and naïve mesenchymal stromal cell-derived extracellular vesicles enhanced E. coli phagocytosis and bacterial killing in human acute monocytic leukemia cell line (THP-1) in vitro (36.9 ± 4, 13.3 ± 8, 0.1 ± 0.01%, respectively; P = 0.0004) compared with vehicle. CONCLUSIONS: Extracellular vesicles from interferon-γ-primed human umbilical cord mesenchymal stromal cells more effectively attenuated E. coli-induced lung injury compared with extracellular vesicles from naïve mesenchymal stromal cells, potentially via enhanced macrophage phagocytosis and killing of E. coli.

Sunitinib induces early histomolecular changes in a subset of renal cancer cells that contribute to resistance.

Sunitinib is the standard-of-care, first-line treatment for advanced renal cell carcinoma (RCC). Characteristics of treatment-resistant RCC have been described; however, complex tumor adaptation mechanisms obstruct the identification of significant operators in resistance. We hypothesized that resistance is a late manifestation of early, treatment-induced histomolecular alterations; therefore, studying early drug response may identify drivers of resistance. We describe an epithelioid RCC growth pattern in RCC xenografts, which emerges in sunitinib-sensitive tumors and is augmented during resistance. This growth modality is molecularly and morphologically related to the RCC spheroids that advance during in vitro treatment. Based on time-lapse microscopy, mRNA and microRNA screening, and tumor behavior-related characteristics, we propose that the spheroid and adherent RCC growth patterns differentially respond to sunitinib. Gene expression analysis indicated that sunitinib promoted spheroid formation, which provided a selective survival advantage under treatment. Functional studies confirm that E-cadherin is a key contributor to the survival of RCC cells under sunitinib treatment. In summary, we suggest that sunitinib-resistant RCC cells exist in treatment-sensitive tumors and are histologically identifiable.-Lichner, Z., Saleeb, R., Butz, H., Ding, Q., Nofech-Mozes, R., Riad, S., Farag, M., Varkouhi, A. K., dos Santos, C. C., Kapus, A., Yousef, G. M. Sunitinib induces early histomolecular changes in a subset of renal cancer cells that contribute to resistance.

MRI-assessed therapeutic effects of locally administered PLGA nanoparticles loaded with anti-inflammatory siRNA in a murine arthritis model.

Rheumatoid arthritis is characterized by systemic inflammation of synovial joints leading to erosion and cartilage destruction. Although efficacious anti-tumor necrosis factor α (TNF-α) biologic therapies exist, there is an unmet medical need for safe and more efficient treatment regimens for disease remission. We evaluated the anti-inflammatory effects of poly(dl-lactide-co-glycolide acid) (PLGA) nanoparticles loaded with small interfering RNA (siRNA) directed against TNF-α in vitro and in vivo. The siRNA-loaded PLGA nanoparticles mediated a dose-dependent TNF-α silencing in lipopolysaccharide-activated RAW 264.7 cells in vitro. The severity of collagen antibody-induced arthritis in DBA/1J mice was assessed by paw scoring and compared to the degree of magnetic resonance imaging (MRI)-quantified joint effusion and bone marrow edema. Two intra-articular treatments per joint with nanoparticles loaded with TNF-α siRNA (1 µg) resulted in a reduction in disease activity, evident by a significant decrease of the paw scores and joint effusions, as compared to treatment with PLGA nanoparticles loaded with non-specific control siRNA, whereas the degree of bone marrow edema in the tibial and femoral head remained unchanged. When the siRNA dose was 5 or 10 µg, there was no difference between the specific and the non-specific siRNA treatment groups. These findings suggest that MRI is a promising method for evaluation of early disease progression and treatment in murine arthritis models. In addition, proper siRNA dosing seems to be important for a positive therapeutic outcome in vivo. However, further studies are needed to fully clarify the mechanism(s) underlying the observed anti-inflammatory effects of the siRNA-loaded nanoparticles.

Comparison of polymeric siRNA nanocarriers in a murine LPS-activated macrophage cell line: gene silencing, toxicity and off-target gene expression.

PURPOSE: Tumor necrosis factor α (TNF-α) plays a key role in the progression of rheumatoid arthritis and is an important target for anti-rheumatic therapies. TNF-α expression can be silenced with small interfering RNA (siRNA), but efficacy is dependent on efficient and safe siRNA delivery vehicles. We aimed to identify polymeric nanocarriers for anti-TNF-α siRNA with optimal efficacy and minimal off-target effects in vitro. METHODS: TNF-α silencing with polymeric siRNA nanocarriers was compared in lipopolysaccharide-activated RAW 264.7 macrophages by real-time reverse transcription (RT)-PCR. Expression of non-target genes involved in inflammation, apoptosis, and cell cycle progression was determined by RT-PCR, toxicity evaluated by propidium iodide and annexin V staining. RESULTS: PAMAM dendrimers (G4 and G7) and dextran nanogels mediated remarkably high concentration-dependent gene silencing and low toxicity; dioleoyltrimethylammoniumpropane-modified poly(DL-lactide-co-glycolide acid) nanoparticles, thiolated, trimethylated chitosan and poly[(2-hydroxypropyl)methacrylamide 1-methyl-2-piperidine methanol] polyplexes were less efficient transfectants. There were minor changes in the regulation of off-target genes, mainly dependent on nanocarrier and siRNA concentration. CONCLUSIONS: Dextran nanogels and PAMAM dendrimers mediated high gene silencing with minor toxicity and off-target transcriptional changes and are therefore expected to be suitable siRNA delivery systems in vivo.

Polyplexes based on cationic polymers with strong nucleic acid binding properties.

Cationic polymers have been studied for nucleic acid delivery both in vitro and in vivo. However, many polymer-based formulations suffer from lack of stability in biologic fluids due to interactions with anionic biomacromolecules such as proteins and polysaccharides. Likely, the stronger the electrostatic interactions between a cationic polymer and nucleic acids, the higher the stability of the polyplexes in biologic fluids will be. To get evidence for this hypothesis, quaternized poly[3,5-bis(dimethylaminomethylene)-p-hydroxyl styrene] (QNPHOS) with two permanently charged cationic sites per monomer unit as well as its block copolymer with PEG were synthesized and compared with the standard transfectant pDMAEMA, in terms of nucleic acid binding strength, gene silencing and transfection activities of the complexes which these polymers form with siRNA and plasmid DNA, respectively. It was shown that siRNA complexes based on QNPHOS and QNPHOS-PEG dissociate in the presence of a fourfold higher heparin concentration than necessary to destabilize pDMAEMA complexes. Under the same conditions, complexes of DNA and QNPHOS or QNPHOS-PEG did not show any dissociation, in contrast to pDMAEMA polyplexes. The DNA polyplexes based on QNPHOS or QNPHOS-PEG did not show transfection activity, which might be ascribed to their high physicochemical stability. On the other hand, siRNA complexes based on QNPHOS and QNPHOS-PEG showed a low cytotoxicity and an improved siRNA delivery and high gene silencing activity, even higher than those based on pDMAEMA. This might be due to the excellent binding characteristics of QNPHOS and QNPHOS-PEG to siRNA which in turn is ascribed to the presence of two permanently charged cationic groups per monomer unit. Based on the results of this study, it is concluded that formation of strong siRNA complexes with polymers containing double charges per monomer is advantageous.

Gene silencing activity of siRNA polyplexes based on biodegradable polymers.

Cationic polymers are used as non-viral vectors for nucleic acid delivery. In this study, two biodegradable cationic polymers were evaluated for the purpose of siRNA delivery: pHPMA-MPPM (poly((2-hydroxypropyl) methacrylamide 1-methyl-2-piperidine methanol)) and TMC (O-methyl-free N,N,N-trimethylated chitosan). The silencing activity and the cellular cytotoxicity of polyplexes based on these biodegradable polymers were compared with those based on non-biodegradable pDMAEMA (poly(2-dimethylamino)ethyl methacrylate) and PEI (polyethylenimine) and with the regularly used lipidic transfection agent Lipofectamine. To promote endosomal escape, either the endosomolytic peptide diINF-7 was added to the formulations or photochemical internalization (PCI) was applied. Incubation of H1299 human lung cancer cells expressing firefly luciferase with polyplexes based on pHPMA-MPPM and TMC showed 30-40% silencing efficiency. This silencing activity was equal to or better than that obtained with the standard transfectants. Under all experimental conditions tested, the cytotoxicity of the biodegradable polymers was low. The application of PCI, as well as the addition of the diINF-7 peptide to the formulations increased their silencing activity up to 70-80%. This demonstrates that pHPMA-MPPM- and TMC-based polyplexes benefit substantially from endosomal escape enhancement. Importantly, the polyplexes retained their silencing activity in the presence of serum, and they showed low cytotoxicity. These biodegradable vectors are therefore attractive systems for further in vivo evaluations.

Gene silencing activity of siRNA polyplexes based on thiolated N,N,N-trimethylated chitosan.

N,N,N-Trimethylated chitosan (TMC) is a biodegradable polymer emerging as a promising nonviral vector for nucleic acid and protein delivery. In the present study, we investigated whether the introduction of thiol groups in TMC enhances the extracellular stability of the complexes based on this polymer and promotes the intracellular release of siRNA. The gene silencing activity and the cellular cytotoxicity of polyplexes based on thiolated TMC were compared with those based on the nonthiolated counterpart and the regularly used lipidic transfection agent Lipofectamine. Incubation of H1299 human lung cancer cells expressing firefly luciferase with siRNA/thiolated TMC polyplexes resulted in 60-80% gene silencing activity, whereas complexes based on nonthiolated TMC showed less silencing (40%). The silencing activity of the complexes based on Lipofectamine 2000 was about 60-70%. Importantly, the TMC-SH polyplexes retained their silencing activity in the presence of hyaluronic acid, while nonthiolated TMC polyplexes hardly showed any silencing activity, demonstrating their stability against competing anionic macromolecules. Under the experimental conditions tested, the cytotoxicity of the thiolated and nonthiolated siRNA complexes was lower than those based on Lipofectamine. Given the good extracellular stability and good silencing activity, it is concluded that polyplexes based on TMC-SH are attractive systems for further in vivo evaluations.

Core-crosslinked polymeric micelles with controlled release of covalently entrapped doxorubicin.

Doxorubicin (DOX) is clinically applied in cancer therapy, but its use is associated with dose limiting severe side effects. Core-crosslinked biodegradable polymeric micelles composed of poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide-lactate] (mPEG-b-p(HPMAm-Lac(n))) diblock copolymers have shown prolonged circulation in the blood stream upon intravenous administration and enhanced tumor accumulation through the enhanced permeation and retention (EPR) effect. However a (physically) entrapped anticancer drug (paclitaxel) was previously shown to be rapidly eliminated from the circulation, likely because the drug was insufficiently retained in the micelles. To fully exploit the EPR effect for drug targeting, a DOX methacrylamide derivative (DOX-MA) was covalently incorporated into the micellar core by free radical polymerization. The structure of the doxorubicin derivative is susceptible to pH-sensitive hydrolysis, enabling controlled release of the drug in acidic conditions (in either the intratumoral environment and/or the endosomal vesicles). 30-40% w/w of the added drug was covalently entrapped, and the micelles with covalently entrapped DOX had an average diameter of 80 nm. The entire drug payload was released within 24 h incubation at pH 5 and 37 degrees C, whereas only around 5% release was observed at pH 7.4. DOX micelles showed higher cytotoxicity in B16F10 and OVCAR-3 cells compared to DOX-MA, likely due to cellular uptake of the micelles via endocytosis and intracellular drug release in the acidic organelles. The micelles showed better anti-tumor activity than free DOX in mice bearing B16F10 melanoma carcinoma. The results presented in this paper show that mPEG-b-p(HPMAm-Lac(n)) polymeric micelles with covalently entrapped doxorubicin is a system highly promising for the targeted delivery of cytostatic agents.