RESUMEN
PURPOSE: To identify a novel, sensitive, nonradioactive leakage assay that can be used in the assessment of retinal vascular permeability in rats and mice. METHODS: Breakdown of the vascular barrier was induced by vascular endothelial growth factor (VEGF), lipopolysaccharide (LPS), or diabetes. Biotinylated bovine serum albumin (bBSA) was administered as a tracer. After perfusion with lactated Ringer's solution, extravasated bBSA was detected with immunoprecipitation and Western blot analysis or sandwich ELISA. The results were then normalized against the final bBSA plasma concentration, the circulation time, and the protein concentration of the tissue. RESULTS: Six hours after VEGF injection, BRB breakdown was quantified in the injected eye and was 2.5-fold higher than in the contralateral phosphate-buffered saline (PBS)-injected eye (n = 6 rats, P < 0.01). Intravitreal LPS injection induced severe inflammation in the directly injected eye and moderate inflammation in the contralateral untreated eye. Leakage was six- and threefold higher, respectively, compared with that in the untreated control animals (n = 5 rats, P < 0.01). Nine-month diabetic rats had a threefold increase in vascular leakage compared with age-matched control animals (n = 6 retinas, P < 0.05). Twenty-four hours after intraperitoneal administration of LPS in mice, the animals showed increased vascular leakage in all tissue organs examined (retina, 1.7-fold; brain, 1.5-fold; and kidney, 1.3-fold). CONCLUSIONS: bBSA can serve as an effective alternative to the current methods used for quantitating vascular leakage and especially the blood-retinal barrier breakdown. It is reasonably easy to perform, low in cost, and adaptable to experiments in mice.
Asunto(s)
Barrera Hematorretinal , Síndrome de Fuga Capilar/diagnóstico , Permeabilidad Capilar , Vasos Retinianos/patología , Albúmina Sérica Bovina , Animales , Biotinilación , Western Blotting , Síndrome de Fuga Capilar/etiología , Diabetes Mellitus Experimental/complicaciones , Ensayo de Inmunoadsorción Enzimática , Inmunoprecipitación , Lipopolisacáridos/toxicidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Endogámicas BN , Vasos Retinianos/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/toxicidadRESUMEN
Mullerian Inhibiting Substance (MIS), a member of the TGF-beta family, causes regression of the Mullerian duct in male embryos, after binding to Mullerian Inhibiting Substance Receptor II (MISRII). It has also been extensively demonstrated that it can inhibit proliferation of various cancer cell lines such as ovarian, prostate, and breast cancer in vitro and in vivo. Hence, the availability of a recombinant, epitope tagged, bioactive MIS is important for the selection of patients for treatment and for probing novel molecular targets for MIS in various tissues. To this end, we have expressed a recombinant, internally FLAG-tagged form of hMIS with the tag (DYKDDDDK) immediately after the cleavage site (427-428) of MIS at the C-terminus with a modified dibasic cleavage motif sequence. We show that this construct results in a highly pure, endogenously processed (cleaved) FLAG MIS, that causes complete regression of the Mullerian Duct in an organ culture assay. In addition, purified FLAG MIS was able to bind and affinity purify both transfected and endogenous MIS type II receptor. The availability of this fully functional, epitope tagged form of MIS should facilitate scale-up for preclinical and clinical use and should also be used for the study of MIS binding proteins and for tracking in pharmacokinetic studies.