Endothelial NOX5 overexpression induces changes in the cardiac gene profile: potential impact in myocardial infarction?

Cardiovascular diseases and the ischemic heart disease specifically constitute the main cause of death worldwide. The ischemic heart disease may lead to myocardial infarction, which in turn triggers numerous mechanisms and pathways involved in cardiac repair and remodeling. Our goal in the present study was to characterize the effect of the NADPH oxidase 5 (NOX5) endothelial expression in healthy and infarcted knock-in mice on diverse signaling pathways. The mechanisms studied in the heart of mice were the redox pathway, metalloproteinases and collagen pathway, signaling factors such as NFκB, AKT or Bcl-2, and adhesion molecules among others. Recent studies support that NOX5 expression in animal models can modify the environment and predisposes organ response to harmful stimuli prior to pathological processes. We found many alterations in the mRNA expression of components involved in cardiac fibrosis as collagen type I or TGF-ß and in key players of cardiac apoptosis such as AKT, Bcl-2, or p53. In the heart of NOX5-expressing mice after chronic myocardial infarction, gene alterations were predominant in the redox pathway (NOX2, NOX4, p22phox, or SOD1), but we also found alterations in VCAM-1 and ß-MHC expression. Our results suggest that NOX5 endothelial expression in mice preconditions the heart, and we propose that NOX5 has a cardioprotective role. The correlation studies performed between echocardiographic parameters and cardiac mRNA expression supported NOX5 protective action.

Structure, regulation, and physiological functions of NADPH oxidase 5 (NOX5).

NOX5 is the last member of the NADPH oxidase (NOXs) family to be identified and presents some specific characteristics differing from the rest of the NOXs. It contains four Ca2+ binding domains at the N-terminus and its activity is regulated by the intracellular concentration of Ca2+. NOX5 generates superoxide (O2•-) using NADPH as a substrate, and it modulates functions related to processes in which reactive oxygen species (ROS) are involved. Those functions appear to be detrimental or beneficial depending on the level of ROS produced. For example, the increase in NOX5 activity is related to the development of various oxidative stress-related pathologies such as cancer, cardiovascular, and renal diseases. In this context, pancreatic expression of NOX5 can negatively alter insulin action in high-fat diet-fed transgenic mice. This is consistent with the idea that the expression of NOX5 tends to increase in response to a stimulus or a stressful situation, generally causing a worsening of the pathology. On the other hand, it has also been suggested that it might have a positive role in preparing the body for metabolic stress, for example, by inducing a protective adipose tissue adaptation to the excess of nutrients supplied by a high-fat diet. In this line, its endothelial overexpression can delay lipid accumulation and insulin resistance development in obese transgenic mice by inducing the secretion of IL-6 followed by the expression of thermogenic and lipolytic genes. However, as NOX5 gene is not present in rodents and human NOX5 protein has not been crystallized, its function is still poorly characterized and further extensive research is required.

Antifibrogenic and apoptotic effects of Ocoxin in cultured rat hepatic stellate cells.

Ocoxin is a nutritional supplement that has been shown to exert antioxidant and immunomodulatory responses in patients with chronic hepatitis C. The present work aimed to determine the effects of Ocoxin on activated hepatic stellate cells (HSC), the cell type mainly responsible for collagen deposition in the fibrotic liver. Ocoxin was found to reduce the survival of a cell line of immortalized non-tumoral rat HSC in a dose-response fashion and to diminish collagen type I levels. This latter effect was observed even at doses not affecting cell survival, pointing to an antifibrogenic action for the supplement. The decrease in viability exerted by Ocoxin on HSC correlated with an increase in histone-associated fragments in the cytoplasm and with increased activity of caspase-3, indicating the induction of apoptosis. To determine the molecular mechanisms mediating Ocoxin-induced apoptosis, the activation of members of the MAPK family was analyzed. Incubation of HSC with Ocoxin caused a transient and dramatic enhancement on ERK, JNK, and p38 MAPK phosphorylation levels. Using specific inhibitors for these enzymes, p38 MAPK was identified as a key mediator of the apoptotic effect of Ocoxin on HSC.

Endothelial Nox5 Expression Modulates Glucose Uptake and Lipid Accumulation in Mice Fed a High-Fat Diet and 3T3-L1 Adipocytes Treated with Glucose and Palmitic Acid.

Obesity is a global health issue associated with insulin resistance and altered lipid homeostasis. It has been described that reactive oxygen species (ROS) derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activity are involved in the development of these pathologies. The present study describes the role of endothelial NOX5 expression over adipose tissue by using two experimental systems: NOX5 conditional knock-in mice fed with a high-fat diet and 3T3-L1 adipocytes cultured with conditioned media of NOX5-expressing endothelial cells previously treated with glucose and palmitic acid. Animals expressing NOX5 presented lower body weight gain and less mesenteric and epididymal adipose mass compared to control mice fed with the same diet. NOX5-expressing mice also showed significantly lower glycaemia and improved insulin-induced glucose uptake. In addition, Glut4 and Caveolin 1 (Cav1) expression were significantly increased in the adipose tissue of these animals. Likewise, 3T3-L1 adipocytes treated with conditioned media from NOX5-expressing endothelial cells, incubated with high glucose and palmitic acid, presented a reduction in lipid accumulation and an increase in glucose uptake. Moreover, a significant increase in the expression of Glut4 and Cav1 was also detected in these cells. Taken together, all these data support that, in response to a highly caloric diet, NOX5 endothelial activity may regulate glucose sensitivity and lipid homeostasis in the adipose tissue.

NADPH Oxidase 5 Induces Changes in the Unfolded Protein Response in Human Aortic Endothelial Cells and in Endothelial-Specific Knock-in Mice.

Oxidative stress constitutes a key molecular mechanism in the development of cardiovascular diseases. A potential relationship between reactive oxygen species (ROS) driven by the NADPH oxidase family (NOX) and the unfolded protein response (UPR) has been postulated. Nevertheless, there is a lack of information about the crosstalk between NOX5 homologue and the UPR in a cardiovascular context. The main aim was to analyze NOX5-mediated ROS effects in the UPR and its importance in cardiovascular diseases. To this effect, we used an adenoviral NOX5-ß overexpression model in human aortic endothelial cells (HAEC) and a conditional endothelial NOX5 knock-in mouse. Using expression arrays, we investigated NOX5-induced genomic changes in HAEC. Compared with the control HAEC, 298 genes were differentially expressed. Gene ontology analysis revealed the activation of numerous cellular routes, the most relevant being the UPR pathway. Using real-time PCR and Western Blot experiments, we confirmed that NOX5 overexpression induced changes in the expression of the UPR components, which were associated with increased apoptosis. Moreover, in endothelial-specific NOX5 knock-in mice, we found changes in the expression of the UPR components genes. In these mice, myocardial infarction was performed by permanent coronary artery ligation; however, NOX5 expression was not associated with differences in the UPR components mRNA levels. In these animals, we found significant associations between the UPR components gene expression and echocardiographic parameters. Our data support the idea that NOX5-derived ROS may modulate the UPR pathway in endothelial cells, which might play a relevant role in cardiac physiology.

Optimization of a GDNF production method based on Semliki Forest virus vector.

Human glial cell line-derived neurotrophic factor (hGDNF) is the most potent dopaminergic factor described so far, and it is therefore considered a promising drug for Parkinson's disease (PD) treatment. However, the production of therapeutic proteins with a high degree of purity and a specific glycosylation pattern is a major challenge that hinders its commercialization. Although a variety of systems can be used for protein production, only a small number of them are suitable to produce clinical-grade proteins. Specifically, the baby hamster kidney cell line (BHK-21) has shown to be an effective system for the expression of high levels of hGDNF, with appropriate post-translational modifications and protein folding. This system, which is based on the electroporation of BHK-21 cells using a Semliki Forest virus (SFV) as expression vector, induces a strong shut-off of host cell protein synthesis that simplify the purification process. However, SFV vector exhibits a temperature-dependent cytopathic effect on host cells, which could limit hGDNF expression. The aim of this study was to improve the expression and purification of hGDNF using a biphasic temperature cultivation protocol that would decrease the cytopathic effect induced by SFV. Here we show that an increase in the temperature from 33°C to 37°C during the "shut-off period", produced a significant improvement in cell survival and hGDNF expression. In consonance, this protocol led to the production of almost 3-fold more hGDNF when compared to the previously described methods. Therefore, a "recovery period" at 37°C before cells are exposed at 33°C is crucial to maintain cell viability and increase hGDNF expression. The protocol described constitutes an efficient and highly scalable method to produce highly pure hGDNF.

Endothelial NOX5 Expression Modulates Thermogenesis and Lipolysis in Mice Fed with a High-Fat Diet and 3T3-L1 Adipocytes through an Interleukin-6 Dependent Mechanism.

Obesity is a global health issue associated with the development of metabolic syndrome, which correlates with insulin resistance, altered lipid homeostasis, and other pathologies. One of the mechanisms involved in the development of these pathologies is the increased production of reactive oxygen species (ROS). One of the main producers of ROS is the family of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, among which NOX5 is the most recently discovered member. The aim of the present work is to describe the effect of endothelial NOX5 expression on neighboring adipose tissue in obesity conditions by using two systems. An in vivo model based on NOX5 conditional knock-in mice fed with a high-fat diet and an in vitro model developed with 3T3-L1 adipocytes cultured with conditioned media of endothelial NOX5-expressing bEnd.3 cells, previously treated with glucose and palmitic acid. Endothelial NOX5 expression promoted the expression and activation of specific markers of thermogenesis and lipolysis in the mesenteric and epididymal fat of those mice fed with a high-fat diet. Additionally, the activation of these processes was derived from an increase in IL-6 production as a result of NOX5 activity. Accordingly, 3T3-L1 adipocytes treated with conditioned media of endothelial NOX5-expressing cells, presented higher expression of thermogenic and lipolytic genes. Moreover, endothelial NOX5-expressing bEnd.3 cells previously treated with glucose and palmitic acid also showed interleukin (IL-6) production. Finally, it seems that the increase in IL-6 stimulated the activation of markers of thermogenesis and lipolysis through phosphorylation of STAT3 and AMPK, respectively. In conclusion, in response to obesogenic conditions, endothelial NOX5 activity could promote thermogenesis and lipolysis in the adipose tissue by regulating IL-6 production.

Induction of Cyclooxygenase-2 by Overexpression of the Human NADPH Oxidase 5 (NOX5) Gene in Aortic Endothelial Cells.

Oxidative stress is a main molecular mechanism that underlies cardiovascular diseases. A close relationship between reactive oxygen species (ROS) derived from NADPH oxidase (NOX) activity and the prostaglandin (PG) biosynthesis pathway has been described. However, little information is available about the interaction between NOX5 homolog-derived ROS and the PG pathway in the cardiovascular context. Our main goal was to characterize NOX5-derived ROS effects in PG homeostasis and their potential relevance in cardiovascular pathologies. For that purpose, two experimental systems were employed: an adenoviral NOX5-ß overexpression model in immortalized human aortic endothelial cells (TeloHAEC) and a chronic infarction in vivo model developed from a conditional endothelial NOX5 knock-in mouse. NOX5 increased cyclooxygenase-2 isoform (COX-2) expression and prostaglandin E2 (PGE2) production through nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in TeloHAEC. Protein kinase C (PKC) activation and intracellular calcium level (Ca++) mobilization increased ROS production and NOX5 overexpression, which promoted a COX-2/PGE2 response in vitro. In the chronic infarction model, mice encoding endothelial NOX5 enhanced the cardiac mRNA expression of COX-2 and PGES, suggesting a COX-2/PGE2 response to NOX5 presence in an ischemic situation. Our data support that NOX5-derived ROS may modulate the COX-2/PGE2 axis in endothelial cells, which might play a relevant role in the pathophysiology of heart infarction.

AGAP2: Modulating TGFß1-Signaling in the Regulation of Liver Fibrosis.

AGAP2 (Arf GAP with GTP-binding protein-like domain, Ankyrin repeat and PH domain 2) isoform 2 is a protein that belongs to the Arf GAP (GTPase activating protein) protein family. These proteins act as GTPase switches for Arfs, which are Ras superfamily members, being therefore involved in signaling regulation. Arf GAP proteins have been shown to participate in several cellular functions including membrane trafficking and actin cytoskeleton remodeling. AGAP2 is a multi-tasking Arf GAP that also presents GTPase activity and is involved in several signaling pathways related with apoptosis, cell survival, migration, and receptor trafficking. The increase of AGAP2 levels is associated with pathologies as cancer and fibrosis. Transforming growth factor beta-1 (TGF-ß1) is the most potent pro-fibrotic cytokine identified to date, currently accepted as the principal mediator of the fibrotic response in liver, lung, and kidney. Recent literature has described that the expression of AGAP2 modulates some of the pro-fibrotic effects described for TGF-ß1 in the liver. The present review is focused on the interrelated molecular effects between AGAP2 and TGFß1 expression, presenting AGAP2 as a new player in the signaling of this pro-fibrotic cytokine, thereby contributing to the progression of hepatic fibrosis.

Role of AGAP2 in the profibrogenic effects induced by TGFß in LX-2 hepatic stellate cells.

Liver damage induces hepatic stellate cells (HSC) activation, characterised by a fibrogenic, proliferative and migratory phenotype. Activated HSC are mainly regulated by transforming growth factor ß 1 (TGFß1), which increases the production of extracellular matrix proteins (e.g. collagen-I) promoting the progression of hepatic fibrosis. AGAP2 (ArfGAP with GTPase domain, ankyrin repeat and PH domain 2) is a GTPase/GTP-activating protein involved in the actin remodelling system and receptor recycling. In the present work the role of AGAP2 in human HSC in response to TGFß1 was investigated. LX-2 HSC were transfected with AGAP2 siRNA and treated with TGFß1. AGAP2 knockdown prevented to some extent the proliferative and migratory TGFß1-induced capacities of LX-2 cells. An array focused on human fibrosis revealed that AGAP2 knockdown partially prevented TGFß1-mediated gene expression of the fibrogenic genes ACTA2, COL1A2, EDN1, INHBE, LOX, PDGFB, TGFΒ12, while favored the expression of CXCR4, IL1A, MMP1, MMP3 and MMP9 genes. Furthermore, TGFß1 induced AGAP2 promoter activation and its protein expression in LX-2. Moreover, AGAP2 protein levels were significantly increased in liver samples from rats with thioacetamide-induced fibrosis. In addition, AGAP2 silencing affected TGFß1-receptor 2 (TGFR2) trafficking in U2OS cells, blocking its effective recycling to the membrane. AGAP2 silencing in LX-2 cells prevented the TGFß1-induced increase of collagen-I protein levels, while its overexpression enhanced collagen-I protein expression in the presence or absence of the cytokine. AGAP2 overexpression also increased focal adhesion kinase (FAK) phosphorylated levels in LX-2 cells. FAK and MEK1 inhibitors prevented the increase of collagen-I expression caused by TGFß1 in LX-2 overexpressing AGAP2. In summary, the present work shows for the first time, that AGAP2 is a potential new target involved in TGFß1 signalling, contributing to the progression of hepatic fibrosis.

NADPH oxidase 5 promotes proliferation and fibrosis in human hepatic stellate cells.

NADPH oxidase (Nox) variants Nox1, Nox2 and Nox4 are implicated in the progression of liver fibrosis. However, the role of Nox5 is not yet known, mainly due to the lack of this enzyme in rat and mouse genomes. Here we describe the expression and functional relevance of Nox5 in the human cell line of hepatic stellate cells (HSC) LX-2. Under basal conditions, three long (Nox5-L: Nox5α, -ß, and -δ) and a short (Nox5-S or Nox5ε) splice variants were detected, which were silenced with specific siRNAs for Nox5. The most abundant isoform was Nox5-S, accounting for more than 90% of Nox5 protein. Overexpression of Nox5ß generated reactive oxygen species (ROS) in the presence of calcium, as judged by the production of hydrogen peroxide, L-012 luminescence and cytochrome c reduction. Nox5ε did not generated ROS under these conditions, and a reduced ROS production was observed when co-expressed with Nox5ß. In contrast, dihydroethidium oxidation was increased by Nox5ß or Nox5ε, suggesting that Nox5ε induced intracellular oxidative stress by an unknown mechanism. Functional studies showed that both Nox5ß and Nox5ε stimulated the proliferation of LX-2 cells and the collagen type I levels, while Nox5 siRNAs inhibited these effects. Interestingly, TGF-ß and angiotensin II upregulated Nox5 expression, which was reduced in cells pre-incubated with catalase. Further studies silencing Nox5 in TGF-ß-treated cells resulted in a reduction of collagen levels via p38 MAPK. Collectively, these results show for the first time that Nox5 can play a relevant role in the proliferation and fibrosis on human HSC.

Brain delivery of microencapsulated GDNF induces functional and structural recovery in parkinsonian monkeys.

Glial cell line-derived neurotrophic factor (GDNF) remains the most potent neurotrophic factor for dopamine neurons. Despite its potential as treatment for Parkinson's disease (PD), its clinical application has been hampered by safety and efficacy concerns associated with GDNF's short in vivo half-life and with significant brain delivery obstacles. Drug formulation systems such as microparticles (MPs) may overcome these issues providing protein protection from degradation and sustained drug release over time. We therefore sought to evaluate the efficacy and safety of GDNF delivered via injectable biodegradable MPs in a clinically relevant model of PD and to investigate the mechanism contributing to their beneficial effects. MPs were injected unilaterally into the putamen of parkinsonian monkeys with severe nigrostriatal degeneration. Notably, a single administration of the microencapsulated neurotrophic factor achieved sustained GDNF levels in the brain, providing motor improvement and dopaminergic function restoration. This was reflected by a bilateral increase in the density of striatal dopaminergic neurons 9 months after treatment. Moreover, GDNF was retrogradely transported to the substantia nigra increasing bilaterally the number of dopaminergic and total neurons, regardless of the severe degeneration. GDNF-MP injection within the putamen elicited no adverse effects such as immunogenicity, cerebellar degeneration or weight loss. MPs are therefore a safe, efficient vehicle for sustained protein delivery to the brain, supporting the therapeutic benefit of GDNF when encapsulated within MPs for brain repair. Overall, these findings constitute important groundwork for GDNF-MP clinical development.

Unfolded protein response induced by Brefeldin A increases collagen type I levels in hepatic stellate cells through an IRE1α, p38 MAPK and Smad-dependent pathway.

Unfolded protein response (UPR) triggered as a consequence of ER stress has been shown to be involved in the development of different pathologies, including fibrotic disorders. In the present paper we explore the role played by UPR on a key fibrogenic parameter in the liver: collagen type I levels in activated hepatic stellate cells (HSC). Using Brefeldin A (BFA) as an ER stress inducer we found that UPR correlated with enhanced mRNA and protein levels of collagen type I in a cell line of immortalized non-tumoral rat HSC. Analysis of the three branches of UPR revealed the activation of IRE1α, PERK and ATF6 in response to BFA, although PERK activation was shown not to be involved in the fibrogenic action of BFA. BFA also activated p38 MAPK in an IRE1α-dependent way and the p38 MAPK inhibitor SB203580 prevented the increase in collagen type I mRNA and protein levels caused by BFA, suggesting the involvement of this kinase on this effect. Analysis of Smad activation showed that phosphorylated nuclear levels of Smad2 and 3 were increased in response to BFA treatment. Inhibition of Smad3 phosphorylation by SIS3 prevented the enhancement of collagen type I levels caused by BFA. Pretreatment with IRE1α and p38 MAPK inhibitors also prevented the increased p-Smad3 accumulation in the nucleus, suggesting an IRE1α-p38 MAPK-Smad pathway to be responsible for the fibrogenic action of BFA on HSC.

Oxidation pathways underlying the pro-oxidant effects of apigenin.

Apigenin, a natural flavone, is emerging as a promising compound for the treatment of several diseases. One of the hallmarks of apigenin is the generation of intracellular reactive oxygen species (ROS), as judged by the oxidation of reduced dichlorofluorescein derivatives seen in many cell types. This study aimed to reveal some mechanisms by which apigenin can be oxidized and how apigenin-derived radicals affect the oxidation of 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein (H(2)DCF), a probe usually employed to detect intracellular ROS. Apigenin induced a rapid oxidation of H(2)DCF in two different immortalized cell lines derived from rat and human hepatic stellate cells. However, apigenin did not generate ROS in these cells, as judged by dihydroethidium oxidation and extracellular hydrogen peroxide production. In cell-free experiments we found that oxidation of apigenin leads to the generation of a phenoxyl radical, which directly oxidizes H(2)DCF with catalytic amounts of hydrogen peroxide. The net balance of the reaction was the oxidation of the probe by molecular oxygen due to redox cycling of apigenin. This flavonoid was also able to deplete NADH and glutathione by a similar mechanism. Interestingly, H(2)DCF oxidation was significantly accelerated by apigenin in the presence of horseradish peroxidase and xanthine oxidase, but not with other enzymes showing peroxidase-like activity, such as cytochrome c or catalase. We conclude that in cells treated with apigenin oxidation of reduced dichlorofluorescein derivatives does not measure intracellular ROS and that pro- and antioxidant effects of flavonoids deduced from these experiments are inconclusive and must be confirmed by other techniques.

Vascular endothelial growth factor-loaded injectable hydrogel enhances plasticity in the injured spinal cord.

We hypothesized that vascular endothelial growth factor (VEGF)-containing hydrogels that gelify in situ after injection into a traumatized spinal cord, could stimulate spinal cord regeneration. Injectable hydrogels composed of 0.5% Pronova UPMVG MVG alginate, supplemented or not with fibrinogen, were used. The addition of fibrinogen to alginate had no effect on cell proliferation in vitro but supported neurite growth ex vivo. When injected into a rat spinal cord in a hemisection model, alginate supplemented with fibrinogen was well tolerated. The release of VEGF that was incorporated into the hydrogel was influenced by the VEGF formulation [encapsulated in microspheres or in nanoparticles or in solution (free)]. A combination of free VEGF and VEGF-loaded nanoparticles was mixed with alginate:fibrinogen and injected into the lesion of the spinal cord. Four weeks post injection, angiogenesis and neurite growth were increased compared to hydrogel alone. The local delivery of VEGF by injectable alginate:fibrinogen-based hydrogel induced some plasticity in the injured spinal cord involving fiber growth into the lesion site.

Injectable alginate hydrogel loaded with GDNF promotes functional recovery in a hemisection model of spinal cord injury.

We hypothesized that local delivery of GDNF in spinal cord lesion via an injectable alginate hydrogel gelifying in situ would support spinal cord plasticity and functional recovery. The GDNF release from the hydrogel was slowed by GDNF encapsulation in microspheres compared to non-formulated GDNF (free GDNF). When injected in a rat spinal cord hemisection model, more neurofilaments were observed in the lesion when the rats were treated with free GDNF-loaded hydrogels. More growing neurites were detected in the tissues surrounding the lesion when the animals were treated with GDNF microsphere-loaded hydrogels. Intense GFAP (astrocytes), low ßIII tubulin (neural cells) and RECA-1 (endothelial cells) stainings were observed for non-treated lesions while GDNF-treated spinal cords presented less GFAP staining and more endothelial and nerve fiber infiltration in the lesion site. The animals treated with free GDNF-loaded hydrogel presented superior functional recovery compared with the animals treated with the GDNF microsphere-loaded hydrogels and non-treated animals.

A simple and efficient method for the production of human glycosylated glial cell line-derived neurotrophic factor using a Semliki Forest virus expression system.

Human glial cell line-derived neurotrophic factor (hGDNF) is a very promising protein for the treatment of Parkinson's disease and other neurodegenerative disorders. The present work describes a quick and simple method to obtain a high amount of purified hGDNF using a mammalian cell-derived system. The method is based on the high expression level provided by a Semliki Forest virus vector and its ability to induce a strong shut-off of host-cell protein synthesis in mammalian cells. As a result, hGDNF is the only protein present in the supernatant and can be efficiently purified by a single chromatographic step. Using this system it was possible to eliminate other secreted proteins from the culture medium, like insulin-like growth factor-5, which are hard to remove using other hGDNF production methods. Purified hGDNF presents a complex glycosylation pattern typical of mammalian expression systems and is biologically active. This protocol could be extended to other secreted proteins and could be easily scaled up for industrial purposes.

PLGA-based nanoparticles: an overview of biomedical applications.

Poly(lactic-co-glycolic acid) (PLGA) is one of the most successfully developed biodegradable polymers. Among the different polymers developed to formulate polymeric nanoparticles, PLGA has attracted considerable attention due to its attractive properties: (i) biodegradability and biocompatibility, (ii) FDA and European Medicine Agency approval in drug delivery systems for parenteral administration, (iii) well described formulations and methods of production adapted to various types of drugs e.g. hydrophilic or hydrophobic small molecules or macromolecules, (iv) protection of drug from degradation, (v) possibility of sustained release, (vi) possibility to modify surface properties to provide stealthness and/or better interaction with biological materials and (vii) possibility to target nanoparticles to specific organs or cells. This review presents why PLGA has been chosen to design nanoparticles as drug delivery systems in various biomedical applications such as vaccination, cancer, inflammation and other diseases. This review focuses on the understanding of specific characteristics exploited by PLGA-based nanoparticles to target a specific organ or tissue or specific cells.

Long-term neuroprotection and neurorestoration by glial cell-derived neurotrophic factor microspheres for the treatment of Parkinson's disease.

BACKGROUND: Glial cell-derived neurotrophic factor is a survival factor for dopaminergic neurons and a promising candidate for the treatment of Parkinson's disease. However, the delivery issue of the protein to the brain still remains unsolved. Our aim was to investigate the effect of long-term delivery of encapsulated glial cell-derived neurotrophic factor within microspheres. METHODS: A single dose of microspheres containing 2.5 µg of glial cell-derived neurotrophic factor was implanted intrastriatally in animals 2 weeks after a 6-hydroxydopamine lesion. RESULTS: The amphetamine test showed a complete behavioral recovery after 16 weeks of treatment, which was maintained until the end of the study (week 30). This effect was accompanied by an increase in dopaminergic striatal terminals and neuroprotection of dopaminergic neurons. CONCLUSIONS: The main achievement was the long-term neurorestoration in parkinsonian animals induced by encapsulated glial cell-derived neurotrophic factor, suggesting that microspheres may be considered as a means to deliver glial cell-derived neurotrophic factor for Parkinson's disease treatment.

Production of highly pure human glycosylated GDNF in a mammalian cell line.

The administration of glial cell line-derived neurotrophic factor (GDNF) has emerged as a promising strategy for the treatment of several diseases of the nervous system as Parkinson's disease, amyotrophic lateral sclerosis, spinal cord injury and nerve regeneration as well as ocular diseases and drug addictions. A procedure for the purification of human recombinant glycosylated GDNF using a mammalian expression system as the source of the protein is discussed in the present paper. The neurotrophic factor was purified using cation exchange chromatography and gel filtration. A human cell line was chosen as the source of therapeutic protein, since a recombinant protein with a structure and glycosylation pattern equivalent to the native form is desirable for its prospective therapeutic utilization. The activity of the highly pure protein obtained was confirmed with a cell-based bioassay. The purified protein is suitable for its in vivo evaluation in animals and for possible subsequent clinical application.