ABSTRACT
BACKGROUND: Leptin, the cytokine produced by white adipose tissue is known to regulate food energy homeostasis through its hypothalamic receptor. In vitro studies have demonstrated that leptin plays a major role in angiogenesis through binding to the receptor Ob-R present on ECs by stimulating and initiating new capillary like structures from ECs. Various in vivo studies indicate that leptin has diverse effect on angiogenesis. A few reports have showed that leptin exerts pro angiogenic effects while some suggested that it has antiangiogenic potential. It is theoretically highly important to understand the effect of leptin on angiogenesis to use as a therapeutic molecule in various angiogenesis related pathological conditions. Chicken chorio allantoic membrane (CAM) on 9th day of incubation was incubated with 1, 3 and 5 µg concentration of HRL for 72 h using gelatin sponge. Images where taken after every 24 h of incubation and analysed with Angioguant software. The treated area was observed under microscope and histological evaluation was performed for the same. Tissue thickness was calculated morphometrically from haematoxylin and eosin stained cross sections. Reverse transcriptase PCR and immunohistochemistry were also performed to study the gene and protein level expression of angiogenic molecules. RESULTS: HRL has the ability to induce new vessel formation at the treated area and growth of the newly formed vessels and cellular morphological changes occur in a dose dependent manner. Increase in the tissue thickness at the treated area is suggestive of initiation of new capillary like structures. Elevated mRNA and protein level expression of VEGF165 and MMP2 along with the activation of ECs as demonstrated by the presence of CD34 expression supports the neovascularization potential of HRL. CONCLUSION: Angiogenic potential of HRL depends on the concentration and time of incubation and is involved in the activation of ECs along with the major interaction of VEGF 165 and MMP2. It is also observed that 3 µg of HRL exhibits maximum angiogenic potential at 72 h of incubation. Thus our data suggest that dose dependent angiogenic potential HRL could provide a novel role in angiogenic dependent therapeutics such as ischemia and wound healing conditions.
Subject(s)
Humans , Animals , Chick Embryo , Zygote , Neovascularization, Physiologic/drug effects , Leptin/administration & dosage , Endothelial Cells/drug effects , Angiogenesis Inducing Agents/administration & dosage , Chorioallantoic Membrane/drug effects , Recombinant Proteins/pharmacology , RNA, Messenger/metabolism , Immunohistochemistry , Gelatinases/metabolism , Antigens, CD34/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Matrix Metalloproteinase 2/metabolism , Vascular Endothelial Growth Factor A/metabolism , Chorioallantoic Membrane/enzymology , Chorioallantoic Membrane/blood supply , Dose-Response Relationship, Drug , MicroscopyABSTRACT
Objective:To explore the purification method of non- affinity chromatography for leptin expressed in Pichia Pastoris.Methods:Ion exchange chromatography (Sepharose Q fast flow) and hydrophobic interaction chromatography (Phenyl Sepharose 6 fast flow ) were used to purify human leptin in pH 7.5. Results: After purification by Q column,the purity of leptin increased from 42.3% to 89.6%.Subsequently, after hydrophobic interaction chromatography ,its purity reached 96.2%. In SDS-PAGE, leptin was shown as one specific band.Conclusion:The human recombinant leptin expressed by Pichia Pastoris yeast can be successfully purified by ion exchange chromatography plus hydrophobic interaction chromatography.