Analysis of local extracellular matrix identifies different aetiologies behind bicuspid and tricuspid aortic valve degeneration and suggests therapies.

Aortic valve degeneration (AVD) is a life-threatening condition that has no medical treatment and lacks individual therapies. Although extensively studied with standard approaches, aetiologies behind AVD are unclear. We compared abundances of extracellular matrix (ECM) proteins from excised valve tissues of 88 patients with isolated AVD of normal tricuspid (TAV) and congenital bicuspid aortic valves (BAV), quantified more than 1400 proteins per ECM sample by mass spectrometry, and demonstrated that local ECM preserves molecular cues of the pathophysiological processes. The BAV ECM showed enrichment with fibrosis markers, namely Tenascin C, Osteoprotegerin, and Thrombospondin-2. The abnormal physical stress on BAV may cause a mechanical injury leading to a continuous Tenascin C-driven presence of myofibroblasts and persistent fibrosis. The TAV ECM exhibited enrichment with Annexin A3 (p = 1.1 × 10-16 and the fold change 6.5) and a significant deficit in proteins involved in high-density lipid metabolism. These results were validated by orthogonal methods. The difference in the ECM landscape suggests distinct aetiologies between AVD of BAV and TAV; warrants different treatments of the patients with BAV and TAV; elucidates the molecular basis of AVD; and implies possible new therapeutic approaches. Our publicly available database (human_avd_ecm.surgsci.uu.se) is a rich source for medical doctors and researchers who are interested in AVD or heart ECM in general. Systematic proteomic analysis of local ECM using the methods described here may facilitate future studies of various tissues and organs in development and disease.

An integrative proteomics method identifies a regulator of translation during stem cell maintenance and differentiation.

Detailed characterization of cell type transitions is essential for cell biology in general and particularly for the development of stem cell-based therapies in regenerative medicine. To systematically study such transitions, we introduce a method that simultaneously measures protein expression and thermal stability changes in cells and provide the web-based visualization tool ProteoTracker. We apply our method to study differences between human pluripotent stem cells and several cell types including their parental cell line and differentiated progeny. We detect alterations of protein properties in numerous cellular pathways and components including ribosome biogenesis and demonstrate that modulation of ribosome maturation through SBDS protein can be helpful for manipulating cell stemness in vitro. Using our integrative proteomics approach and the web-based tool, we uncover a molecular basis for the uncoupling of robust transcription from parsimonious translation in stem cells and propose a method for maintaining pluripotency in vitro.

Redox responsive Pluronic micelle mediated delivery of functional siRNA: a modular nano-assembly for targeted delivery.

There is an unmet need to develop strategies that allow site-specific delivery of short interfering RNA (siRNA) without any associated toxicity. To address this challenge, we have developed a novel siRNA delivery platform using chemically modified pluronic F108 as an amphiphilic polymer with a releasable bioactive disulfide functionality. The micelles exhibited thermoresponsive properties and showed a hydrodynamic size of ∼291 nm in DLS and ∼200-250 nm in SEM at 37 °C. The grafting of free disulfide pyridyl groups enhanced the transfection efficiency and was successfully demonstrated in human colon carcinoma (HCT116; 88%) and glioma cell lines (U87; 90%), non-cancerous human dermal fibroblast (HDF; 90%) cells as well as in mouse embryonic stem (mES; 54%) cells. To demonstrate the versatility of our modular nanocarrier design, we conjugated the MDGI receptor targeting COOP peptide on the particle surface that allowed the targeted delivery of the cargo molecules to human patent-derived primary BT-13 gliospheres. Transfection experiments with this design resulted in ∼65% silencing of STAT3 mRNA in BT-13 gliospheres, while only ∼20% of gene silencing was observed in the absence of the peptide. We believe that our delivery method solves current problems related to the targeted delivery of RNAi drugs for potential in vivo applications.

Pluronic Micelle-Mediated Tissue Factor Silencing Enhances Hemocompatibility, Stemness, Differentiation Potential, and Paracrine Signaling of Mesenchymal Stem Cells.

Mesenchymal stem/stromal cells (MSCs) evoke great excitement for treating different human diseases due to their ability to home inflamed tissues, suppress inflammation, and promote tissue regeneration. Despite great promises, clinical trial results are disappointing as allotransplantation of MSCs trigger thrombotic activity and are damaged by the complement system, compromising their survival and function. To overcome this, a new strategy is presented by the silencing of tissue factor (TF), a transmembrane protein that mediates procoagulant activity. Novel Pluronic-based micelles are designed with the pendant pyridyl disulfide group, which are used to conjugate TF-targeting siRNA by the thiol-exchange reaction. This nanocarrier design effectively delivered the payload to MSCs resulting in ∼72% TF knockdown (KD) without significant cytotoxicity. Hematological evaluation of MSCs and TF-KD MSCs in an ex vivo human whole blood model revealed a significant reduction in an instant-blood-mediated-inflammatory reaction as evidenced by reduced platelet aggregation (93% of free platelets in the TF-KD group, compared to 22% in untreated bone marrow-derived MSCs) and thrombin-antithrombin complex formation. Effective TF silencing induced higher MSC differentiation in osteogenic and adipogenic media and showed stronger paracrine suppression of proinflammatory cytokines in macrophages and higher stimulation in the presence of endotoxins. Thus, TF silencing can produce functional cells with higher fidelity, efficacy, and functions.

Effect of the Addition Frequency of 5-Azacytidine in Both Micro- and Macroscale Cultures.

INTRODUCTION: Human mesenchymal stem cells (hMSCs) have a great clinical potential for tissue regeneration purposes due to its multilineage capability. Previous studies have reported that a single addition of 5-azacytidine (5-AzaC) causes the differentiation of hMSCs towards a myocardial lineage. The aim of this work was to evaluate the effect of 5-AzaC addition frequency on hMSCs priming (i.e., indicating an early genetic differentiation) using two culture environments. METHODS: hMSCs were supplemented with 5-AzaC while cultured in well plates and in microfluidic chips. The impact of 5-AzaC concentration (10 and 20 µM) and addition frequency (once, daily or continuously), as well as of culture period (2 or 5 days) on the genetic upregulation of PPARγ (adipocytes), PAX3 (myoblasts), SOX9 (chondrocytes) and RUNX2 (osteoblasts) was evaluated. RESULTS: Daily delivering 5-AzaC caused a higher upregulation of PPARγ, SOX9 and RUNX2 in comparison to a single dose delivery, both under static well plates and dynamic microfluidic cultures. A particularly high gene expression of PPARγ (tenfold-change) could indicate priming of hMSCs towards adipocytes. CONCLUSIONS: Both macro- and microscale cultures provided results with similar trends, where addition frequency of 5-AzaC was a crucial factor to upregulate several genes. Microfluidics technology was proven to be a suitable platform for the continuous delivery of a drug and could be used for screening purposes in tissue engineering research.

First Aldol Cross-Linked Hyaluronic Acid Hydrogel: Fast and Hydrolytically Stable Hydrogel with Tissue Adhesive Properties.

Currently, there are limited approaches to tailor 3D scaffolds cross-linked with a stable covalent C-C bond that does not require any catalysts or initiators. We present here the first hydrogels employing aldol condensation chemistry that exhibit exceptional physicochemical properties. We investigated the aldol-cross-linking chemistry using two types of aldehyde-modified hyaluronic acid (HA) derivatives, namely, an enolizable HA-aldehyde (HA-Eal) and a non-enolizable HA-aldehyde (HA-Nal). Hydrogels formed using HA-Eal demonstrate inferior cross-linking efficiency (due to intramolecular loop formation), when compared with hydrogels formed by mixing HA-Eal and HA-NaI leading to a cross-aldol product. The change in mechanical properties as a result of cross-linking at different pH values is determined using rheological measurements and is interpreted in terms of molecular weight between cross-links (Mc). The novel HA cross-aldol hydrogel demonstrate excellent hydrolytic stability and favorable mechanical properties but allow hyaluronidase-mediated enzymatic degradation. Interestingly, residual aldehyde functionality within the aldol product rendered the tissue-adhesive properties by bonding two bone tissues. The aldehyde functionality also facilitated facile post-synthetic modifications with nucleophilic reagents. Finally, we demonstrate that the novel hydrogel is biocompatible with encapsulated stem cells that show a linear rate of expansion in our 3-6 days of study.

Correction: 4'-Guanidinium-modified siRNA: a molecular tool to control RNAi activity through RISC priming and selective antisense strand loading.

Correction for '4'-Guanidinium-modified siRNA: a molecular tool to control RNAi activity through RISC priming and selective antisense strand loading' by Ganesh N. Nawale et al., Chem. Commun., 2019, 55, 9112-9115.

4'-Guanidinium-modified siRNA: a molecular tool to control RNAi activity through RISC priming and selective antisense strand loading.

We designed novel 4'-C-guanidinocarbohydrazidomethyl-5-methyl uridine (GMU) modified small interfering RNA (siRNA) and evaluated its biophysical and biochemical properties. Incorporation of GMU units significantly increased the thermodynamic stability as well as the enzymatic stability against nucleases in human serum. A gene silencing experiment indicated that GMU modfied siRNA (siRNA6) resulted in ≈4.9-fold more efficient knockdown than unmodified siRNA.

Synthetic Design of Asymmetric miRNA with an Engineered 3' Overhang to Improve Strand Selection.

We developed a novel miRNA design that significantly improves strand selection within the RISC complex by engineering the 3' end by adding extra nucleotides. Addition of seven nucleotides at the 3' ends of the miR or miR* strand resulted in a thermodynamic asymmetry at either of the two ends, which resulted in selective RISC recruitment, as demonstrated by a stem-loop PCR experiment. Such selective recruitment was also corroborated at the protein level by western blot analysis. To investigate the functional effect because of selective recruitment, we performed apoptosis and metastasis studies using human colon carcinoma cells (HCT116) and human osteosarcoma cells (MG63). These experiments indicated that recruitment of the miR strand is responsible for inducing apoptosis and inhibiting the invasiveness of cancer cells. Recruitment of the miR* strand, on the other hand, had the opposite effect. To the best of our knowledge, our strand engineering strategy is the first report of improved strand selection of a desired miRNA strand by RISC without using any chemical modifications or mismatches. We believe that such structural modifications of miR34a could mitigate some of the off-target effects of miRNA therapy and would also allow a better understanding of sequence-specific gene regulation. Such a design could also be adapted to other miRNAs to enhance their therapeutic potential.

An Unexpected Role of Hyaluronic Acid in Trafficking siRNA Across the Cellular Barrier: The First Biomimetic, Anionic, Non-Viral Transfection Method.

Circulating nucleic acids, such as short interfering RNA (siRNA), regulate many biological processes; however, the mechanism by which these molecules enter the cell is poorly understood. The role of extracellular-matrix-derived polymers in binding siRNAs and trafficking them across the plasma membrane is reported. Thermal melting, dynamic light scattering, scanning electron microscopy, and computational analysis indicate that hyaluronic acid can stabilize siRNA via hydrogen bonding and Van der Waals interactions. This stabilization facilitated HA size- and concentration-dependent gene silencing in a CD44-positive human osteosarcoma cell line (MG-63) and in human mesenchymal stromal cells (hMSCs). This native HA-based siRNA transfection represents the first report on an anionic, non-viral delivery method that resulted in approximately 60 % gene knockdown in both cell types tested, which correlated with a reduction in translation levels.

Innovative Strategy for 3D Transfection of Primary Human Stem Cells with BMP-2 Expressing Plasmid DNA: A Clinically Translatable Strategy for Ex Vivo Gene Therapy.

Ex vivo gene therapy offers enormous potential for cell-based therapies, however, cumbersome in vitro cell culture conditions have limited its use in clinical practice. We have optimized an innovative strategy for the transient transfection of bone morphogenetic protein-2 (BMP-2) expressing plasmids in suspended human stem cells within 5-min that enables efficient loading of the transfected cells into a 3D hydrogel system. Such a short incubation time for lipid-based DNA nanoparticles (lipoplexes) reduces cytotoxicity and at the same time reduces the processing time for cells to be transplanted. The encapsulated human mesenchymal stromal/stem cells (hMSCs) transfected with BMP-2 plasmid demonstrated high expression of an osteogenic transcription factor, namely RUNX2, but not the chondrogenic factor (SOX9), within the first three days. This activation was also reflected in the 7-day and 21-day experiment, which clearly indicated the induction of osteogenesis but not chondrogenesis. We believe our transient transfection method demonstrated in primary MSCs can be adapted for other therapeutic genes for different cell-based therapeutic applications.

Saline Accelerates Oxime Reaction with Aldehyde and Keto Substrates at Physiological pH.

We have discovered a simple and versatile reaction condition for oxime mediated bioconjugation reaction that could be adapted for both aldehyde and keto substrates. We found that saline accelerated the oxime kinetics in a concentration-dependent manner under physiological conditions. The reaction mechanism is validated by computational studies, and the versatility of the reaction is demonstrated by cell-surface labeling experiments. Saline offers an efficient and non-toxic catalytic option for performing the bioorthogonal-coupling reaction of biomolecules at the physiological pH. This saline mediated bioconjugation reaction represents the most biofriendly, mild and versatile approach for conjugating sensitive biomolecules and does not require any extensive purification step.

Fast and Efficient Transfection of Mouse Embryonic Stem Cells Using Non-Viral Reagents.

Reliable and efficient DNA and RNA transfection methods are required when studying the role of individual genes in mouse pluripotent stem cells. However, these cells usually grow in tight clusters and are therefore more difficult to transfect than many other cell lines. We have found that transfection is especially challenging when mouse embryonic stem (mES) cells are cultured in the newly described 2i medium, which is based on two chemical inhibitors of differentiation pathways. In the present study we have performed a side-by-side comparison of commercially available, non-viral transfection reagents with regard to their ability to deliver plasmid DNA and siRNA into adherent and/or trypsinized mES cells cultured in 2i medium, assessing transfection rates, plasmid gene expression, siRNA mediated knockdown of Oct4 and viability. Finally, we present a fast and efficient method for transfection of trypsinized mES cells using the liposomal-based Lipofectamine 2000. With only a five-minute long transfection time we obtained at least 85 % transfected cells with 80 % maintained viability. Moreover, this protocol saves up to a day of experimental time since the cells are in suspension at the time of transfection, which allows for immediately re-plating into the appropriate format. This fast, simplified and highly efficient transfection method will be valuable for both basic research and high-throughput applications.

PCB153, TCDD and estradiol compromise the benzo[a]pyrene-induced p53-response via FoxO3a.

TCDD, polychlorinated biphenyls (PCB) and polycyclic aromatic hydrocarbons (PAH) coexist in the environment. However, there are few studies on combined effects of these compounds. We have studied the effect of TCDD, PCB153 and estradiol on p53 signaling induced by PAHs. We show that all three compounds amplified the accumulation of nuclear p53, elicited by benzo[a]pyrene (BaP) or dibenzo[al]pyrene (DBP). This effect was associated with an attenuated PAH-induced apoptosis and with decreased levels of phosphorylated FoxO3a Thr32. Thr32 phosphorylation of FoxO3a may promote a translocation of FoxO3a-p53 complex from nucleus to the cytoplasm, and the role of FoxO3a dephosphorylation was further studied. We found that inhibition of PP2A phosphatase restored levels of phosphorylated FoxO3a, led to cytosolic translocation of p53, and activated BaP-induced p53-mediated apoptosis. These results were confirmed by silencing FoxO3a with siRNA or by inhibiting 14-3-3 protein; also these treatments trapped BaP-induced p53 in the nucleus. Our data indicate interplay between p53, FoxO3a and 14-3-3 leading to an attenuated BaP induced apoptosis in cells co-exposed to TCDD, PCB 153 or estradiol.

Exocrine pancreatic carcinogenesis and autotaxin expression.

Exocrine pancreatic cancer is an aggressive disease with an exceptionally high mortality rate. Genetic analysis suggests a causative role for environmental factors, but consistent epidemiological support is scarce and no biomarkers for monitoring the effects of chemical pancreatic carcinogens are available. With the objective to identify common traits for chemicals inducing pancreatic tumors we studied the National Toxicology Program (NTP) bioassay database. We found that male rats were affected more often than female rats and identified eight chemicals that induced exocrine pancreatic tumors in males only. For a hypothesis generating process we used a text mining tool to analyse published literature for suggested mode of actions (MOA). The resulting MOA analysis suggested inflammatory responses as common feature. In cell studies we found that all the chemicals increased protein levels of the inflammatory protein autotaxin (ATX) in Panc-1, MIA PaCa-2 or Capan-2 cells. Induction of MMP-9 and increased invasive migration were also frequent effects, consistent with ATX activation. Testosterone has previously been implicated in pancreatic carcinogenesis and we found that it increased ATX levels. Our data show that ATX is a target for chemicals inducing pancreatic tumors in rats. Several lines of evidence implicate ATX and its product lysophosphatidic acid in human pancreatic cancer. Mechanisms of action may include stimulated invasive growth and metastasis. ATX may interact with hormones or onco- or suppressor-genes often deregulated in exocrine pancreatic cancer. Our data suggest that ATX is a target for chemicals promoting pancreatic tumor development.

Gender differences in chemical carcinogenesis in National Toxicology Program 2-year bioassays.

Differences in cancer incidences between men and women are often explained by either differences in environmental exposures or by influences of sex hormones. However, there are few studies on intrinsic gender differences in susceptibility to chemical carcinogens. We have analyzed the National Toxicology Program (NTP) database for sex differences in rat responses to chemical carcinogens. We found that the odds that male rat bioassays were assigned a higher level of evidence than female rat bioassays was 1.69 (p < .001). Of 278 carcinogenic chemicals in the database, 201 (72%) exhibited statistical gender differences (p ≤ .05) in at least one nonreproductive organ. One hundred thirty of these 201 chemicals induced gender-specific tumors in male rats and 59 in female rats. Sixty-eight chemicals induced tumors in males but no tumors in females. Less than one third (i.e., 19 chemicals) induced tumors in females but not males. Male-specific tumors included pancreatic and skin tumors, and female-specific tumors included lung tumors. For some tumor sites, these differences in gender susceptibility can be associated with literature data on sex hormone receptor expression. In conclusion, gender-specific tumors were common. The male dominance is in line with recent human data, and the male susceptibility to carcinogens should be further studied.

Purinergic receptor-mediated rapid depletion of nuclear phosphorylated Akt depends on pleckstrin homology domain leucine-rich repeat phosphatase, calcineurin, protein phosphatase 2A, and PTEN phosphatases.

Akt is an important oncoprotein, and data suggest a critical role for nuclear Akt in cancer development. We have previously described a rapid (3-5 min) and P2X7-dependent depletion of nuclear phosphorylated Akt (pAkt) and effects on downstream targets, and here we studied mechanisms behind the pAkt depletion. We show that cholesterol-lowering drugs, statins, or extracellular ATP, induced a complex and coordinated response in insulin-stimulated A549 cells leading to depletion of nuclear pAkt. It involved protein/lipid phosphatases PTEN, pleckstrin homology domain leucine-rich repeat phosphatase (PHLPP1 and -2), protein phosphatase 2A (PP2A), and calcineurin. We employed immunocytology, immunoprecipitation, and proximity ligation assay techniques and show that PHLPP and calcineurin translocated to the nucleus and formed complexes with Akt within 3 min. Also PTEN translocated to the nucleus and then co-localized with pAkt close to the nuclear membrane. An inhibitor of the scaffolding immunophilin FK506-binding protein 51 (FKBP51) and calcineurin, FK506, prevented depletion of nuclear pAkt. Furthermore, okadaic acid, an inhibitor of PP2A, prevented the nuclear pAkt depletion. Chemical inhibition and siRNA indicated that PHLPP, PP2A, and PTEN were required for a robust depletion of nuclear pAkt, and in prostate cancer cells lacking PTEN, transfection of PTEN restored the statin-induced pAkt depletion. The activation of protein and lipid phosphatases was paralleled by a rapid proliferating cell nuclear antigen (PCNA) translocation to the nucleus, a PCNA-p21(cip1) complex formation, and cyclin D1 degradation. We conclude that these effects reflect a signaling pathway for rapid depletion of pAkt that may stop the cell cycle.