Heparin Sodium Salt Research Articles

Addressing bioreactor hiPSC aggregate stability, maintenance and scaleup challenges using a design of experiment approach

BackgroundStem cell-derived therapies hold the potential for treatment of regenerative clinical indications. Static culture has a limited ability to scale up thus restricting its use. Suspension culturing can be used to produce target cells in large quantities, but also presents challenges related to stress and aggregation stability.MethodsUtilizing a design of experiments (DoE) approach in vertical wheel bioreactors, we evaluated media additives that have versatile properties. The additives evaluated are Heparin sodium salt (HS), polyethylene glycol (PEG), poly (vinyl alcohol) (PVA), Pluronic F68 and dextran sulfate (DS). Multiple response variables were chosen to assess cell growth, pluripotency maintenance and aggregate stability in response to the additive inputs, and mathematical models were generated and tuned for maximal predictive power.ResultsExpansion of iPSCs using 100 ml vertical wheel bioreactor assay for 4 days on 19 different media combinations resulted in models that can optimize pluripotency, stability, and expansion. The expansion optimization resulted in the combination of PA, PVA and PEG with E8. This mixture resulted in an expansion doubling time that was 40% shorter than that of E8 alone. Pluripotency optimizer highlighted the importance of adding 1% PEG to the E8 medium. Aggregate stability optimization that minimizes aggregate fusion in 3D culture indicated that the interaction of both Heparin and PEG can limit aggregation as well as increase the maintenance capacity and expansion of hiPSCs, suggesting that controlling fusion is a critical parameter for expansion and maintenance. Validation of optimized solution on two cell lines in bioreactors with decreased speed of 40 RPM, showed consistency and prolonged control over aggregates that have high frequency of pluripotency markers of OCT4 and SOX2 (> 90%). A doubling time of around 1–1.4 days was maintained after passaging as clumps in the optimized medium. Controlling aggregate fusion allowed for a decrease in bioreactor speed and therefore shear stress exerted on the cells in a large-scale expansion.ConclusionThis study resulted in a control of aggregate size within suspension cultures, while informing about concomitant state control of the iPSC state. Wider application of this approach can address media optimization complexity and bioreactor scale-up challenges.

Human adenovirus type 3 restores pharmacologically inhibited exosomal cargo in lung carcinoma cells.

Introduction: Drug repurposing is fast growing and becoming an attractive approach for identifying novel targets, such as exosomes for cancer and antiviral therapy. Exosomes are a specialized class of extracellular vesicles that serve as functional mediators in intercellular communication and signaling that are important in normal physiological functions. A continuously growing body of evidence has established a correlation between the abnormal release of exosomes with various viral disease pathologies including cancer. Cells that are virus-infected release exosomes known to influence the process via the loading and transfer of viral components, such as miRNA, small (s) RNA, DNA, and proteins. Inhibition of exosome release may abate the spread and severity of viral infection, thus making exosomes an attractive target for antiviral therapies. We previously demonstrated the pharmacological inhibition of exosomes. Methods: Herein, we used a cell-based assay to determine the effect of Human adenovirus type 3 (HAdV3) on the exosome inhibition process by azole and Heparin derivatives. HAdV3-infected cells were treated with two concentrations of each inhibitor at different time points. Results: HAdV3 activities led to increased total sRNA, DNA, and exosome particle concentrations via particle tracking in the presence of Climbazole and Heparin relative to uninfected exosomes. In addition, there was an increased expression of classical markers such as ALG-2 interacting protein X (ALIX), and tetraspanin (CD63), (p < 0.05) and upregulated transcription factor interferon regulatory factor (IRF) 8 in the presence of HAdV3 after 24hours (h) of treatment. Whereas higher concentrations of Climbazole and Heparin sodium salt were found to inhibit total exosome protein (p < 0.001) and exo-RNA (p < 0.01) content even in the presence of HAdV3 relative to infected exosomes only. Activities of HAdV3 in the presence of selected inhibitors resulted in the positive regulation of exosome related DNA damage/repair signaling proteins. Blocking exosome secretion partially obstructed viral entry. Immunological studies revealed that HAdV3 fiber protein levels in A549 cells were reduced at all concentrations of Climbazole and Heparin and both multiplicities of infections (p < 0.001). Discussion: Our findings suggest that while HAdV may bolster inhibited exosome content and release when modulating certain activities of the endosomal pathway mediators, HAdV entry might be constrained by the activities of these pharmacological agents.

Heparan sulfate is the attachment factor associated with channel catfish virus infection on host cells.

Channel catfish virus (CCV; family Alloherpesviridae) infects channel catfish, causing great harm to aquaculture fisheries and economic development. Attachment is the first step in viral infection and relies on the interaction of virions with components of the extracellular matrix (ECM). The present study aimed to explored the role of the main three ECM components in CCV attachment. Western blotting and quantitative real-time PCR analysis showed that neither collagen nor hyaluronic acid treatments had significant effects on CCV attachment. When exogenous heparin was used as a competitive inhibitor, the adhesion of heparin sodium salt to CCV was dose-dependent. When the concentration of heparin sodium salt was 10 mg/mL, the inhibitory effect on CCV infection of channel catfish ovary (CCO/BB) cells was more than 90%. Heparinase I could significantly prevent CCV attachment by digesting heparan sulfate on the cell surface, and both heparin sodium salt and heparinase I could dose-dependently reduce CCV titers, suggesting that heparin plays an important role in CCV attachment. In addition, the binding experiments between heparin-agarose beads and virions showed that CCV virions could specifically bind to heparin in a dose-dependent manner. The above results suggested that heparan sulfate might be an attachment factor involved in CCV infection of CCO/BB cells. These results increase our understand of the attachment mechanism of CCV and lay the foundation for further research on antiviral drugs.

Periprocedural management of sclerotherapy in patients with telangiectasias of the lower extremities

Objective — to reveal the effect of certain medicinal and non­medicinal factors that can be applied before and after the sclerotherapy procedure on the final result of the treatment. Materials and methods. The study included 104 patients aged 18 to 60 years who underwent sclerotherapy for the treatment of telangiectasias and reticular varicosities of the lower extremities. Patients were divided into subgroups depending on the appointment of phlebotonics and external means after the procedure. Results were evaluated 1, 3, and 6 months after treatment. Results and discussion. According to the self-assessment of patients, the best cosmetic result was achieved in the subgroup that used phlebotonics in combination with external therapy with heparin sodium gel, compared to the subgroups in which phlebotonics were not prescribed (p &lt; 0.01). In the subgroup where only phlebotonics were used, the result was also better compared to the subgroups in which phlebotonics were not prescribed (p = 0.03; p &lt; 0.01). Self-administration of sodium heparin gel did not provide a statistically significant improvement when comparing the subgroups (p = 0.26). No statistically significant difference in disease recurrence was found between the subgroups (p = 0.729). Conclusions. Ultrasound Doppler mapping with a detailed definition of the feeding sources of the reticular veins and telangiectasias allows determining the optimal stages of treatment to prevent the recurrence of the disease. The appointment of phlebotonics in the post­procedure period has a positive effect on the cosmetic result of sclerotherapy, especially in combination with external therapy with heparin sodium salt gel (p &lt; 0.01). The use of phlebotonics and gel with heparin sodium salt does not affect the number of relapses of the disease (p = 0,729), which is probably due to the technical impossibility of detecting feeding sources in the pre­procedural period.

Role of Heparin Sodium Salt in the Modulation of Human Umbilical Cord-Derived Mesenchymal Stem Cell Differentiation

Introduction: The present study aims to investigate the role of low molecular weight compound heparin sodium salt (HSS) to control the differentiation of the human umbilical cord (UC) derived mesenchymal stem cells (MSCs) through possible interaction with WWTR1 protein. Materials and Methods: In order to carry out this study, the human UC-derived stem cells were isolated and characterized by stem cell specific markers and the effect of HSS was studied by altering the phenotypes of MSCs. An Insilco approach was employed to reveal the structural determination of the ligand, the WWTR1 protein binding site and to predict the strength of the interaction. After HSS treatment, WWTR1, Oct4, nanog, SOX9 gene expressions were studied using real-time polymerase chain reaction (PCR). Cell staining was performed using alizarin red to confirm the formation of osteocytes. Results: Mineralization indicated by osteocytes was confirmed using alizarin red after the treatment of HSS. Post, HSS treatment, OCT4, Nanog, RUNX2, COL1A1 and WWTR1 gene expressions were positively modulated. Heparin treatment of MSCs lead to the up regulation of WWTR1 along with the down regulation of stemness markers Oct4 and Nanog expression. In silico studies also predicted the possible interaction of WWTR1 with HS. Results indicated that Amino acid residues ASP57, GLN83, GLN109, THR135, and TYR141 came up as a prominent interaction centre; ASP57, GLN83 and THR135 recorded the highest interaction energy – while ASP57 mostly participated in an electrostatic interaction. Conclusions: To conclude, it can be stated that heparin can possibly interact with WWTR1 along with having the capability to direct cells towards osteogenic lineages.

Immunogenicity of murine mPEG-red blood cells and the risk of anti-PEG antibodies in human blood donors.

The immunocamouflage of non-ABO blood group antigens by membrane-grafted methoxypoly(ethylene glycol) (mPEG) may attenuate the risk of red blood cell (RBC) alloimmunization. However, concerns have been raised over the immunogenic risk of PEG and PEG-RBCs. To assess this risk, murine and human studies were performed. Mice were exposed to soluble PEG prior to, or between, multiple transfusions (∼60-day intervals) of control or mPEG-RBCs, and cell survival was determined by flow cytometry. In some studies, the control and mPEG-RBC groups were reversed after one or more transfusions. Furthermore, human blood donors and commercial intravenous immunoglobulin products were examined to detect anti-PEG antibodies and to assess the risk for false positives. Naïve mice receiving chronic mPEG-RBC transfusions had normal RBC survival curves with no evidence of anti-PEG antibodies. Similarly, challenge with soluble PEG did not elicit anti-PEG antibodies in mice. Studies in humans revealed no evidence of a high prevalence of anti-PEG antibodies in either blood donors or commercial intravenous immunoglobulin. However, by use of the methods employed by studies identifying high levels of anti-PEG antibodies, a significant level (∼15%) of "false positives" were detected in commercial antibodies of known (non-PEG) specificities. These findings suggest that methodologic problems yielded a high rate of false positives in these earlier studies. These data continue to support the clinical utility of cellular PEGylation and the low immunogenic risk of grafted mPEG.

In vitro production of bovine blastocyst with oocyte collected from abattoir ovary

This study was designed to adopt in vitro embryo production (IVP) protocol using abattoir ovary. Ovaries were collected from local abattoir; cumulus-oocyte-complexes (COCs) were aspirated from 3 to 8 mm diameter follicles using a 10 ml disposable syringe attached with a 21G needle. The COCs were selected based on morphological characteristics and selected COCs were transferred into in vitro maturation (IVM) medium for 22 to 24 hrs. The matured COC were fertilized in vitro (IVF) using fresh semen capacitated through incubation with heparin sodium salt (20 ?g/ml). After IVF, the presumed denuded zygotes were cultured in in vitro culture medium-I (IVC-I) for 3 days. After 3 days, the ?8 cell embryos were transferred into IVC-II medium until day 8 of embryonic development (day 0: day of IVF). Cleavage and blastocyst development rates were evaluated at day 3 and day 7. The maturation rates of COC were examined through detection of first polar body and cumulus cell expansion. Results showed that 74.16 ± 5.49% of the total immature COCs were matured as detected by the presence of first polar body. The diameter of matured COC was 2.21 times higher than that of the immature COC. Moreover, about 64.30 ± 6.71% COC showed full expansion of their cumulus cell. The cleavage rate of presumed zygotes was 62.05 ± 7.07%. Among the cleaved embryos, 26.67 ± 11.78%, 10.84 ± 6.13%, 22.51 ± 9.57% and 39.98 ± 5.25% embryos were at 2-, 4-, 8- and 16 to 32-cell embryonic stages, respectively at day 3. Blastocyst development rates were 14.95 ± 4.39%. This study inferred that the BLRI adopted culture system supports development of bovine embryo in vitro.Bang. J. Anim. Sci. 2016. 45 (1): 31-35

C-Terminal Amino Acids 471-507 of Avian Hepatitis E Virus Capsid Protein Are Crucial for Binding to Avian and Human Cells.

The infection of chickens with avian Hepatitis E virus (avian HEV) can be asymptomatic or induces clinical signs characterized by increased mortality and decreased egg production in adult birds. Due to the lack of an efficient cell culture system for avian HEV, the interaction between virus and host cells is still barely understood. In this study, four truncated avian HEV capsid proteins (ORF2-1 – ORF2-4) with an identical 338aa deletion at the N-terminus and gradual deletions from 0, 42, 99 and 136aa at the C-terminus, respectively, were expressed and used to map the possible binding site within avian HEV capsid protein. Results from the binding assay showed that three truncated capsid proteins attached to avian LMH cells, but did not penetrate into cells. However, the shortest construct, ORF2-4, lost the capability of binding to cells suggesting that the presence of amino acids 471 to 507 of the capsid protein is crucial for the attachment. The construct ORF2-3 (aa339-507) was used to study the potential binding of avian HEV capsid protein to human and other avian species. It could be demonstrated that ORF2-3 was capable of binding to QT-35 cells from Japanese quail and human HepG2 cells but failed to bind to P815 cells. Additionally, chicken serum raised against ORF2-3 successfully blocked the binding to LMH cells. Treatment with heparin sodium salt or sodium chlorate significantly reduced binding of ORF2-3 to LMH cells. However, heparinase II treatment of LMH cells had no effect on binding of the ORF2-3 construct, suggesting a possible distinct attachment mechanism of avian as compared to human HEV. For the first time, interactions between avian HEV capsid protein and host cells were investigated demonstrating that aa471 to 507 of the capsid protein are needed to facilitate interaction with different kind of cells from different species.

Competition Between Tamm‐Horsfall Protein and Heparin in Ligand Interactions

Tamm‐Horsfall protein (THP), also called uromodulin, is an 86kD acidic, single‐chain, glycoprotein produced in the kidney and excreted into the urine. THP binds various molecules of the immune system including immunoglobulins (Ig), complement 1q (C1q), and factor H (fH). Heparin is a negatively‐charged, sulfated polysaccharide that binds to a wide range of proteins, including C1q, fH, antithrombin III (ATIII), basic fibroblast growth factor (FGF2), and interleukin‐10 (IL‐10). The purpose of this study was to determine if heparin competed with THP for binding to proteins that interact with both THP and heparin (i.e. C1q and fH), and to screen other heparin‐binding proteins for previously unidentified interactions with THP. Dot blots were performed by immobilizing 1 μg of C1q, fH, and bovine serum albumin (BSA) to 4 strips of nitrocellulose (NC). One NC strip was stained with amido black and the other 3 NC strips were incubated with either THP (10 μg/ml), THP (10 μg/ml)/heparin (20 μg/ml) (sodium salt from porcine intestine), or no THP/heparin, before incubation with sheep anti‐THP antiserum and then donkey anti‐sheep IgG ‐ HRP. These blots showed that THP bound strongly to C1q and fH, and that this binding was inhibited by heparin. Heparin also inhibited the background binding of the antisera to C1q. ELISAs were used to quantify the ability of heparin to inhibit C1q (5 μg/ml) binding to immobilized THP (40 μg/ml). A steep inhibition curve was obtained, with no inhibition of C1q binding to immobilized THP occurring with 0.02 units/ml heparin, 50% inhibition of binding occurring with between 0.04 and 0.05 units heparin/ml, and 90% inhibition occurring with 0.1 unit heparin/ml (170 units/mg heparin). Control wells showed that heparin's inhibition of C1q binding to THP was not due to inhibition by heparin of the antisera's binding to C1q. Additionally, dot blots were used to evaluate binding of soluble THP to ATIII, IL‐10, and FGF2, three of the many proteins that heparin has been shown to bind. These blots demonstrated that THP bound to FGF2 and IL‐10, and this interaction was blocked by the addition of heparin. THP did not bind ATIII. In summary, heparin inhibited THP's binding to NC‐immobilized C1q and fH, as well as inhibiting immobilized THP's binding to soluble C1q in ELISAs. Protein‐protein interactions between THP and FGF2 and IL‐10, that were sensitive to heparin inhibition, were discovered. Heparin and THP coexist in the urine, especially after intravenous heparin administration or when heparin is instilled into the bladder as a therapy for interstitial nephritis. Mast cells are prominent in the wall of the urinary bladder in interstitial cystitis, so competition of heparin and THP for binding to other proteins may be important in this disease. Further studies to assess the physiological significance in THP/heparin competition for binding to various proteins is warranted.Support or Funding InformationFunded by the PNWU Research Seed Program.

Bimolecular based heparin and self-assembling hydrogel for tissue engineering applications

One major goal of tissue engineering is to develop new biomaterials that are similar structurally and functionally to the extracellular matrix (ECM) to mimic natural cell environments. Recently, different types of biomaterials have been developed for tissue engineering applications. Among them, self-assembling peptides are attractive candidates to create artificial cellular niches, because their nanoscale network and biomechanical properties are similar to those of the natural ECM. Here, we describe the development of a new biomaterial for tissue engineering composed by a simple combination of the self-assembling peptide RAD16-I and heparin sodium salt. As a consequence of the presence of heparin moieties the material acquired enhances the capacity of specific binding and release of growth factors (GFs) with heparin binding affinity such as VEGF165. Promising results were obtained in the vascular tissue engineering area, where the new composite material supported the development of tubular-like structures within a three dimensional (3D) culture model. Moreover, the new scaffold enhances the cell survival and chondrogenic commitment of adipose-derived stem cells (ADSC). Interestingly, the expression of specific markers of mature cartilage tissue including collagen type II was confirmed by western blot and real-time PCR. Furthermore, positive staining for proteoglycans (PGs) indicated the synthesis of cartilage tissue ECM components. Finally, the constructs did not mineralize and exhibited mechanical properties of a tissue undergoing chondrogenesis. Altogether, these results suggest that the new composite is a promising “easy to prepare” material for different reparative and regenerative applications.

Glycopolymer Polyelectrolyte Multilayers Composed of Heparin and Maltose‐Modified Poly(ethylene imine) as a Strong/Weak Polyelectrolyte System for Future Drug Delivery Coatings: Influence of pH and Sugar Architecture on Growth of Multilayers and Multilayer Swelling and Stability

Establishing highly sophisticated polymer films for delivery systems in a biological environment and bioanalytical tasks, the formation, thickness, swelling behavior, and (physiological) stability of highly biocompatible polyelectrolyte multilayers (PEMs) are described. These PEMs are composed of the very weak polycation maltose‐modified hyperbranched poly(ethylene imine) (PEI‐Mal) and the strong polyanion heparin sodium salt (HE−Na+) deposited on Si wafer substrates . Two different glyco architectures for PEI‐Mal are used, characterized by two different degrees of maltose decoration on a PEI scaffold. Using two pH‐dependent deposition approaches for optimizing the (physiological) PEM stability and swelling, PEMs are characterized by (in situ) ellipsometry, atomic force microscopy (AFM), and (in situ) attenuated total reflection‐Fourier‐transform infrared (ATR‐FTIR). Thus, PEMs reveal significantly different thicknesses, growth mechanisms (linear versus exponential), and swelling behavior in dependence of both the polycation architectures and the deposition protocol. These PEMs will allow the study of their complexation and release properties as preswollen PEMs against anionic drug molecules, especially under physiological conditions in the future. image

Controlled release studies of antimalarial 1, 3, 5-trisubstituted-2-pyrazolines from biocompatible chitosan–heparin Layer-by-Layer (LbL) self assembled thin films

Herein we report the in-vitro controlled release properties of 1, 3, 5-trisubstituted-2-pyrazolines through Layer-by-Layer (LbL) self assembled thin films fabricated from chitosan and heparin sodium salt as biocompatible polyelectrolytes. This study was carried out as a preliminary step towards the applicability of LbL technique in prophylactic drug delivery of antimalarial drugs. The growth of LbL self assembly was monitored by UV–Visible spectrophotometry and Quartz Crystal Microbalance (QCM). The loading as well as in-vitro release studies (in phosphate buffer saline at pH 7.4) were carried out using UV–Visible spectroscopy. Three compounds having good antimalarial activity were tested and the release rate was found inversely proportional to the hydrophobicity of the drug. Pzln-4 has shown best release among all the three compounds (up to 780min) followed by Pzln-5 and Pzln-8. The release trend was that of a fast release up to first 2h followed by a steady release. Kinetic fitting of the data confirmed the process of drug release followed a pseudo second order kinetics (R2≥0.99). A large value of rate constant (k) revealed a faster release. Pzln-4 has shown smallest value of k corresponding to slowest release among all the three compounds.

218 IMPROVEMENT OF INTRACYTOPLASMIC SPERM INJECTION MEDIATED TRANSGENESIS (TM-INTRACYTOPLASMIC SPERM INJECTION) USING BULL SPERM PRETREATED WITH HEPARIN AND GLUTATHIONE

TM-intracytoplasmic sperm injection (ICSI) was demonstrated to be an effective technique for the production of transgenic animals. However, this method has not been widely applied for transgenesis in cattle, because of the low embryo developmental rates. This problem may be related to the incomplete sperm decondensation and subsequent pronuclei formation that occurs in cattle after ICSI (Malcuit et al. 2006 Reprod. Fertil. Dev. 18, 39–51). Delgado et al. showed that pretreatment with heparin-sodium salt combined with reduced glutathione (Hep-GSH) could improve bull sperm decondensation (2001 Archives of Andrology 47, 47–58). The objective of this work was to test the use of pretreated sperm with Hep-GSH for TM-ICSI, because an improvement of male pronucleus formation could cause an increase on the frequency of exogenous DNA integration. To this aim, cumulus-oocyte complexes were collected from slaughtered cow ovaries and in vitro matured for 21 h. Frozen sperm from a bull that was previously determined to produce low developmental rates post ICSI and IVF was used. It was thawed and washed twice by centrifuging at 390 × g for 10 min. After that, sperm were incubated with Tris medium supplemented with 80 μM Hep and 15 mM GSH for 20 h. After washing, semen was co-incubated with 50 ng μL–1 of pCX-EGFP plasmid for 5 min on ice and used for ICSI (Hep-GSH ICSI group). An ICSI control group was injected with semen not treated with Hep-GSH. Sham controls were injected with 50 ng μL–1 of pCX-EGFP. Haploid and diploid parthenogenetic controls were also included (Haplo PA and Diplo PA groups). Oocytes were activated by a 4 min exposure to 5 μM ionomycin, placed on TCM-199 for 3 h, and treated with 1.9 mM DMAP for 3 h; Diplo PA were immediately exposed to DMAP after ionomycin treatment. Embryos were cultured in SOF medium. Cleavage and blastocyst rates were evaluated on Days 2 and 7 post ICSI, respectively. Expression of egfp was assayed at Day 4 and at the blastocyst stage. Results: Hep-GSH ICSI group showed higher cleavage rates than ICSI control (68.5%, n = 89 v. 35%, n = 60), and lower than Sham, Diplo PA, and Haplo Pa groups (94% n = 50, 95.1% n = 61, and 85.1% n = 47, respectively; Fisher's exact test, P ≤ 0.05). Although blastocyst rates from ICSI groups did not differ from Haplo PA (21.2%) and Sham groups (8%), Hep-GSH ICSI produced higher rates than ICSI control (19.1 v. 5%). The higher blastocyst rates were observed for Diplo PA (47.5%; P ≤ 0.05). Transgene expression levels at Day 4 were higher for both Hep-GSH ICSI and ICSI control than for Sham control (24.7 and 11.7% v. 0%, respectively; P ≤ 0.05). Rates of egfp expressing blastocysts/injected oocytes were significantly higher for Hep-GSH ICSI than for ICSI and Sham control groups (13.5 v. 1.7 and 0%, respectively; P ≤ 0.05). Conclusions: Pretreatment of bull sperm with Hep-GSH can increase blastocyst rates after ICSI, even when low quality semen is used. Additionally, the employment of Hep-GSH treatment increased rates of transgene expressing blastocysts. It could be a useful strategy for massively implementing TM-ICSI in bovine, for the production of transgenic animals.

Engineering strategies to control vascular endothelial growth factor stability and levels in a collagen matrix for angiogenesis: The role of heparin sodium salt and the PLGA-based microsphere approach

New vessel formation is the result of the complex orchestration of various elements, such as cells, signalling molecules and extracellular matrix (ECM). In order to establish the suitable conditions for an effective cell response, the influence of vascular endothelial growth factor (VEGF) complexation with heparin sodium salt (Hp) on its pro-angiogenic activity has been evaluated by an in vitro capillary-like tube formation assay. VEGF with or without Hp was embedded into collagen gels, and the activated matrices were characterized in terms of VEGF activity and release kinetics. Taking into account the crucial role of Hp in VEGF stability and activity, VEGF/Hp complex was then encapsulated into microspheres based on poly(lactide-co-glycolide) (PLGA), and microsphere properties, VEGF/Hp release kinetics and VEGF in vitro activity over time were evaluated. Integrated microsphere/collagen matrices were developed in order to provide a continuous release of active VEGF/Hp inside the matrix but also a VEGF gradient at the boundary, which is an essential condition for endothelial cell attraction and scaffold invasion. The results confirmed a strong influence of Hp on VEGF configuration and, consequently, on its activity, while the encapsulation of VEGF/Hp complex in PLGA-microspheres guaranteed a sustained release of active VEGF for more than 30days. This paper confirms the importance of VEGF stability and signal presentation to cells for an effective proangiogenic activity and highlights how the combination of two stabilizing approaches, namely VEGF/Hp complexation and entrapment within PLGA-based microspheres, may be a very effective strategy to achieve this goal.

Assembly, Purification, and Pre-steady-state Kinetic Analysis of Active RNA-dependent RNA Polymerase Elongation Complex

NS5B is the RNA-dependent RNA polymerase responsible for replicating hepatitis C virus (HCV) genomic RNA. Despite more than a decade of work, the formation of a highly active NS5B polymerase·RNA complex suitable for mechanistic and structural studies has remained elusive. Here, we report that through a novel way of optimizing initiation conditions, we were able to generate a productive NS5B·primer·template elongation complex stalled after formation of a 9-nucleotide primer. In contrast to previous reports of very low proportions of active NS5B, we observed that under optimized conditions up to 65% of NS5B could be converted into active elongation complexes. The elongation complex was extremely stable, allowing purification away from excess nucleotide and abortive initiation products so that the purified complex was suitable for pre-steady-state kinetic analyses of polymerase activity. Single turnover kinetic studies showed that CTP is incorporated with apparent K(d) and k(pol) values of 39 ± 3 μM and 16 ± 1 s(-1), respectively, giving a specificity constant of k(pol)/K(d) of 0.41 μM(-1) s(-1). The kinetics of multiple nucleotide incorporation during processive elongation also were determined. This work establishes a novel way to generate a highly active elongation complex of the medically important NS5B polymerase for structural and functional studies.

Fibroblast Growth Factor Receptor-3 (FGFR-3) Regulates Expression of Paneth Cell Lineage-specific Genes in Intestinal Epithelial Cells through both TCF4/β-Catenin-dependent and -independent Signaling Pathways

Fibroblast growth factor receptor-3 (FGFR-3) expression in the developing intestine is restricted to the undifferentiated epithelial cells within the lower portion of the crypt. We previously showed that mice lacking functional FGFR-3 have a significant decrease in the number of Paneth cells in the small intestine. Here, we used Caco2 cells to investigate whether FGFR-3 signaling can directly modulate expression of Paneth cell differentiation markers through its effects on TCF4/β-catenin or through other signaling pathways downstream of this receptor. Caco2 cells treated with FGFR-3 ligands or expressing FGFR-3(K650E), a constitutively active mutant, resulted in a significantly increased expression of genes characteristic of mature Paneth cells, including human α-defensins 5 and 6 (HD5 and HD6) and Paneth cell lysozyme, whereas enterocytic differentiation markers were reduced. Activation of FGFR-3 signaling sustained high levels of β-catenin mRNA expression, leading to increased TCF4/β-catenin-regulated transcriptional activity in Caco2 cells. Sustained activity of the TCF4/β-catenin pathway was required for the induction of Paneth cell markers. Activation of the MAPK pathway by FGFR-3 is also required for the induction of Paneth cell markers in addition to and independent of the effect of FGFR-3 on TCF4/β-catenin activity. These studies suggest that coordinate activation of multiple independent signaling pathways downstream of FGFR-3 is involved in regulation of Paneth cell differentiation.

Hyaluronic acid/Chitosan nanoparticles as delivery vehicles for VEGF and PDGF-BB

The development of a vascular network in tissue-engineered constructs is a fundamental bottleneck of bioregenerative medicine, particularly when the size of the implant exceeds a certain limit given by diffusion lengths and/or if the host tissue shows a very active metabolism. One of the approaches to achieve the vascularization of tissue constructs is generating a sustained release of proangiogenic factors from the ischemic site. This work describes the formation and characterization of hyaluronic acid-chitosan (HA/CS) nanoparticles for the delivery of two pro-angiogenic growth factors: vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF-BB). These nanoparticles were prepared by an ionic gelification technique, and different formulations were developed by encapsulating the growth factors in association with two stabilizing agents: bovine serum albumin or heparin sodium salt. These carriers were characterized with regard to their physicochemical properties, their stability in biological media, and their cytotoxicity in the C3a hepatoma cell line. The results show that nanoparticles around 200 nm can be prepared by this method. HA/CS nanoparticles were stable when incubated in EMEM cell culture medium or in water at 37°C for 24 h. Cell culture tests confirmed that HA/CS nanoparticles are not cytotoxic within the concentration range used for growth factor delivery. Moreover, HA/CS nanoparticles were able to entrap efficiently both growth factors, reaching association values of 94% and 54% for VEGF and PDGF, respectively. In vitro release studies confirm that PDGF-BB is released from HA/CS nanoparticles in a sustained manner over ∼ 1 week. On the other hand, VEGF is completely released within the first 24 h.

Nanoparticles Based on PLGA:Poloxamer Blends for the Delivery of Proangiogenic Growth Factors

New blood vessel formation is a critical requirement for treating many vascular and ischemia related diseases, as well as for many tissue engineering applications. Angiogenesis and vasculogenesis, in fact, represent crucial processes for the functional regeneration of complex tissues through tissue engineering strategies. Several growth factors (GFs) and signaling molecules involved in blood vessels formation have been identified, but their application to the clinical setting is still strongly limited by their extremely short half-life in the body. To overcome these limitations, we have developed a new injectable controlled release device based on polymeric nanoparticles for the delivery of two natural proangiogenic GFs: platelet derived growth factor (PDGF-BB) and fibroblast growth factor (FGF-2). The nanoparticle system was prepared by a modified solvent diffusion technique, encapsulating the GF both in presence and in the absence of two stabilizing agents: bovine serum albumin (BSA) and heparin sodium salt (Hp). The developed nanocarriers were characterized for morphology, size, encapsulation efficiency, release kinetics in vitro and GF activity in cell cultures. The results have indicated that the coencapsulation of stabilizing agents can preserve the GF active structure and, in addition, increase their encapsulation efficiency into nanoparticles. Through this optimization process, we were able to raise the encapsulation efficiency of FGF-2 to 63%, and that of PDGF-BB to 87%. These PLGA:poloxamer blend nanoparticles loaded with GFs were able to release PDGF-BB and FGF-2 in a sustained fashion for more than a month. This work also confirms other positive features of PLGA:poloxamer nanoparticles. Namely, they are able to maintain their stability in simulated biological medium, and they are also nontoxic to cell culture models. Incubation of nanoparticles loaded with FGF-2 or PDGF-BB with endothelial cell culture models has confirmed that GFs are released in a bioactive form. Altogether, these results underline the interest of PLGA:poloxamer nanoparticles for the controlled delivery of GFs and substantiate their potential for the treatment of ischemic diseases and for tissue engineering applications.

Enrichment of (8,4) Single‐Walled Carbon Nanotubes Through Coextraction with Heparin

Heparin sodium salt is investigated as a dispersant for dispersing single-walled carbon nanotubes (SWNTs). Photoluminescence excitation (PLE) spectroscopy is used for identification and abundance estimation of the chiral species. It is found that heparin sodium salt preferentially disperses larger-diameter Hipco SWNTs. When used to disperse CoMoCAT nanotube samples, heparin has a strong preference for (8,4) tubes, which have larger diameter than the predominant (6,5) in pristine CoMoCAT samples. PLE intensity due to (8,4) tubes increases from 7% to 60% of the total after threefold extractions. Computer modeling verifies that the complex of (8,4) SWNTs and heparin has the lowest binding energy amongst the four semiconducting species present in CoMoCAT. Network field-effect transistors are successfully made with CoMoCAT/heparin and CoMoCAT/sodium dodecylbenzene sulfonate (SDBS)-heparin (x3), confirming the easy removability of heparin.

Chondroitin 4-O-Sulfotransferase-1 Modulates Wnt-3a Signaling through Control of E Disaccharide Expression of Chondroitin Sulfate

Wnt-3a is a ligand that activates the beta-catenin-dependent pathway in Wnt signaling, which is implicated in numerous physiological events such as morphogenesis. So far, heparan sulfate (HS) proteoglycans have been highlighted as a low affinity receptor for morphogens containing Wnts. Here we show the importance of chondroitin sulfate (CS) proteoglycans in the efficient signaling of Wnt-3a and the structural features of CS required for the regulation of Wnt-3a signaling. Wnt-3a signaling was depressed in a mouse L cell mutant, called sog9, which is defective in the EXT1 gene encoding the HS-synthesizing enzyme and the chondroitin 4-O-sulfotransferase (C4ST-1) gene compared with parental L cells. The transfection of sog9 cells with C4ST-1 resulted in the recovery of Wnt-3a signaling, whereas the expression of EXT1 in sog9 cells could not restore Wnt-3a signaling. In addition, the expression level of introduced C4ST-1 correlated with the recovery of Wnt-3a signaling accompanied by the increased expression of the E disaccharide unit of CS. Interestingly, molecular interaction analyses using Biacore revealed that squid CS-E (rich in the E disaccharide unit) bound strongly to Wnt-3a (K(d)=13.2 nm) to the same extent as heparin from bovine lung (K(d)=8.43 nm). In contrast, other CS isoforms as well as HS isolated from bovine kidney showed little binding activity to Wnt-3a. Moreover, exogenously added CS-E potently inhibited the accumulation of beta-catenin induced by Wnt-3a. These results suggest that CS-E-like structures synthesized by C4ST-1 participate in Wnt-3a signaling and modulate the physiological events caused by Wnt-3a signals.