ABSTRACT
The vascular endothelial growth factor (VEGF) is an essential factor to pathologic angiogenesis. Disruption of VEGF/VEGF receptor interaction in cancer patients inhibits the development of new and pre-existing tumor blood vessels. Consequently, VEGF becomes an important therapeutic target for handling solid tumors. In this work, human VEGF was produced in the culture supernatant of SiHa cells transduced with a replication-defective adenoviral vector (pAdhVEGF121) encoding this molecule. The 35 kDa VEGF121 homodimer was obtained from clarified culture media as a glycosylated protein. VEGF121 expression levels were strictly dependent on the adenoviral viral load used. VEGF121 was produced with purity over 98% after a single step chromatography by immobilized metal affinity chromatography. Additionally, VEGF121 binds Bevacizumab antibody with a KD of 7 nM. Biological characterization by mitogenic assay in HUVEC and ECV-304 cells showed that VEGF121 stimulates cell proliferation in a dose-dependent manner in both cells. Finally, the neovascularization activity of VEGF121 was demonstrated by vascular permeability assays in matrigel plug-bearing mice, showing significantly increased vasculature leakage after treatment with VEGF121. Consequently, transduction of SiHa cells with adenovirus is a suitable alternative for manufacture heterologous proteins of therapeutic interest.
Subject(s)
Vascular Endothelial Growth Factor A/isolation & purification , Vascular Endothelial Growth Factor A/pharmacology , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , MiceABSTRACT
Erythropoietin is a glycoproteic hormone that regulates hematopoiesis by acting on its specific receptor (EpoR). The expression of EpoR in the central nervous system (CNS) suggests a role for this hormone in the brain. Recently, we developed a new Epo variant without hematopoietic activity called EpoL, which showed marked neuroprotective effects against oxidative stress in brain ischemia related models. In this study, we have evaluated the neuroprotective effects of EpoL against oxidative stress induced by chronic treatment with Aß. Our results show that EpoL was neuroprotective against Aß-induced toxicity by a mechanism that implicates EpoR, reduction in reactive oxygen species, and reduction in astrogliosis. Furthermore, EpoL treatment improved calcium handling and SV2 levels. Interestingly, the neuroprotective effect of EpoL against oxidative stress induced by chronic Aß treatment was achieved at a concentration 10 times lower than that of Epo. In conclusion, EpoL, a new variant of Epo without hematopoietic activity, is of potential interest for the treatment of diseases related to oxidative stress in the CNS such as Alzheimer disease.
Subject(s)
Erythropoietin/pharmacology , Neuroprotective Agents/pharmacology , Oxidative Stress/drug effects , Peptide Fragments/pharmacology , Recombinant Proteins/pharmacology , Amyloid beta-Peptides/metabolism , Amyloid beta-Peptides/pharmacology , Animals , Calcium Signaling , Cell Line, Tumor , Cell Survival/drug effects , Erythropoietin/genetics , Erythropoietin/isolation & purification , Goats , Milk , Neuroprotective Agents/isolation & purification , Peptide Fragments/chemistry , Protein Multimerization , Reactive Oxygen Species/metabolism , Receptors, Erythropoietin/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purificationABSTRACT
Subunit recombinant vaccines against classical swine fever virus (CSFV) are a promising alternative to overcome practical and biosafety issues with inactivated vaccines. One of the strategies in evaluation under field conditions is the use of a new marker E2-based vaccine produced in the milk of adenovirally transduced goats. Previously we had demonstrated the efficacy of this antigen, which conferred early protection and long-lasting immunity in swine against CSFV infection. Here, we have used a simpler downstream process to obtain and formulate the recombinant E2 glycoprotein expressed in the mammary gland. The expression levels reached approximately 1.7 mg/ml, and instead of chromatographic separation of the antigen, we utilized a clarification process that eliminates the fat content, retains a minor amount of caseins, and includes an adenoviral inactivation step that improves the biosafety of the final formulation. In a vaccination and challenge experiment in swine, different doses of the E2 antigen contained within the clarified whey generated an effective immune response of neutralizing antibodies that protected all of the animals against a lethal challenge with CSFV. During the immunization and after challenge, the swine were monitored for adverse reactions related to the vaccine or symptoms of CSF, respectively. No adverse reactions or clinical signs of disease were observed in vaccinated animals, in which no replication of CSFV could be detected after challenge. Overall, we consider that the simplicity of the procedures proposed here is a further step toward the introduction and implementation of a commercial subunit vaccine against CSF.
