Η ΒΙΟΣΥΝΘΕΣΗ ΤΩΝ ΒΑΣΙΚΩΝ ΜΕΜΒΡΑΝΩΝ ΩΣ ΒΙΟΧΗΜΙΚΟΣ ΔΕΙΚΤΗΣ ΤΗΣ ΑΓΓΕΙΟΓΕΝΕΣΗΣ

Abstract

THE PRESENT THESIS CONCERNS THE DEVELOPMENT OF A NEW METHOD FOR ASSESSING ANGIOGENESIS. THE SYSTEM CHOSEN IS THE CHORIOALLANTOIC MEMBRANE (CAM) OF THE CHICK EMBRYO, AND THE METHOD IS BASED ON THE RATE OF BASEMENT MEMBRANE (BM) BIOSYNTHESIS DURING EMBRYOGENESIS. MORPHOLOGICAL EVALUATION OF ANGIOGENESIS WAS ACHIEVED BY COUNTING VASCULAR DENSITY OF THE CAM FROM DAY 7 TO DAY 20 OF EGG INCUBATION.THE RATE OF BM BIOSYNTHESIS WAS MONITORED BY MEASURING THE INCORPORATION OF [U-14C]-PROLINE, AS PROLINE OR HYDROXYPROLINE, IN COLLAGEN TYPE IV, WHICH IS THE BASIC CONSTITUENT OF BM. COLLAGEN TYPE IV SYNTHESIS OF THE NEWLY FORMED BM WAS ESTIMATED EITHER BY MEASURING [U-14C]- HYDROXYPROLINE, OR BY MEASURING COLLAGENOUS PROTEIN DEGRADATION PRODUCTS, FOLLOWING DIGESTION WITH CLOSTRIDIUM HISTOLYTICUM COLLAGENASE. THE RATE OF COLLAGENOUS PROTEIN BIOSYNTHESIS WAS MAXIMUM BETWEEN DAYS 8 TO 10 OF CHICK EMBRYO DEVELOPEMENT WHICH CORRESPONDED WITH MAXIMUM ANGIOGENESIS, AS INDICATED BY MORPHOLOGICAL EVALUATION OF THE VASCULAR DENSITY. AT DAY 10 THE RATE OF COLLAGENOUS PROTEIN BIOSYNTHESIS WAS 11-FOLD HIGHER THAN THAT AT DAY 15, WHEN ANGIOGENESIS HAD REACHED A PLATEAU. THE COLLAGENOUS PROTEIN FORMED BY CAM CO-ELUTES ON SDS-ARAGOSE CHROMATOGRAPHY WITH THE COLLAGENOUS COMPONENT OF [3H]-ACETYLATED- MEMBRANE FROM BOVINE LENS CAPSULE. IT WAS ALSO SHOWN THAT COMPOUNDS (E.G. GPA-1734) WHICH HAVE BEEN REPORTED TO INHIBIT BM COLLAGEN BIOSYNTHESIS IN OTHER SYSTEMS (E.G. PARIETAL YOLK SAC OF THE RAT), CAUSE ABOUT 80% REDUCTION IN COLLAGENOUS PROTEIN SYNTHESIS OF CAM. THESE FINDINGS INDICATE THAT MOST OF THE COLLAGENOUS PROTEIN SYNTHESIZED BY CAM WAS BM COLLAGEN. USING THE ABOVE IN VITRO SYSTEM OF CAM IT WAS ESTABLISHED THAT SEVERAL COMPOUNDS WHICH INHIBIT BM BIOSYNTHESIS, BIOCHEMICALLY, CAN ALSO PREVENT ANGIOGENESIS AS EVIDENCED BY MORPHOLOGICAL OBSERVATIONS. IN CONCLUSION, THE PROPOSED METHOD OF MONITORING BM BIOSYNTHESIS IS A RELATIVELY SIMPLE, RELIABLE AND QUANTITATIVE BIOCHEMICAL METHOD FOR ASSESSING INHIBITORS OF BM BIOSYNTHESIS. ALTHOUGH INHIBITIONOF BM BIOSYNTHESIS IT IS NOT UNIQUE FOR INHIBITING ANGIOGENESIS, NEVERTHELESS IS AN EFFECTIVE TARGET FOR ANTIANGIOGENIC AGENTS. THUS, THE PROPOSED METHOD OFFERS A CONVENIENT SCREENING TEST FOR COMPOUNDS WITH ANTIANGIOGENIC PROPERTIES WITH CONSIDERABLE THERAPEUTIC USEFULNESS.

ΣΤΗΝ ΕΡΓΑΣΙΑ ΑΥΤΗ ΜΕΛΕΤΗΘΗΚΕ Η ΔΗΜΙΟΥΡΓΙΑ ΑΓΓΕΙΩΝ ΣΤΟ ΕΜΒΡΥΟΝΙΚΟ ΣΥΣΤΗΜΑ ΤΗΣ ΧΟΡΙΟΑΛΛΑΝΤΟΙΚΗΣ ΜΕΜΒΡΑΝΗΣ (CAM) ΓΟΝΙΜΟΠΟΙΗΜΕΝΟΥ ΑΥΓΟΥ ΚΟΤΟΠΟΥΛΟΥ, ΜΕ ΒΑΣΗ ΤΟ ΡΥΘΜΟ ΒΙΟΣΥΝΘΕΣΗΣ ΤΩΝ ΒΑΣΙΚΩΝ ΜΕΜΒΡΑΝΩΝ (ΒΜ) ΚΑΤΑ ΤΑ ΔΙΑΦΟΡΑ ΣΤΑΔΙΑ ΑΝΑΠΤΥΞΗΣ ΤΩΝ ΕΜΒΡΥΩΝ. Η ΜΟΡΦΟΛΟΓΙΚΗ ΑΞΙΟΛΟΓΗΣΗ ΤΗΣ ΑΓΓΕΙΟΓΕΝΕΣΗΣ ΕΓΙΝΕ ΠΡΟΣΔΙΟΡΙΖΟΝΤΑΣ ΤΗΝ ΠΥΚΝΟΤΗΤΑ ΤΩΝ ΑΓΓΕΙΩΝ ΤΗΣ CAM, ΑΠΟ ΤΗΝ 7Η ΕΩΣ ΤΗΝ 20Η ΗΜΕΡΑ ΕΠΩΑΣΗΣ ΤΩΝ ΑΥΓΩΝ. ΟΡΥΘΜΟΣ ΒΙΟΣΥΝΘΕΣΗΣ ΤΩΝ ΒΜ ΥΠΟΛΟΓΙΣΘΗΚΕ ΜΕ ΤΗΝ ΕΝΣΩΜΑΤΩΣΗ (U-14C)-ΠΡΟΛΙΝΗΣ ΣΤΟ ΒΑΣΙΚΟ ΣΥΣΤΑΤΙΚΟ ΤΩΝ ΒΜ, ΤΟ ΚΟΛΛΑΓΟΝΟ ΤΥΠΟΥ IV. Η ΕΝΣΩΜΑΤΩΣΗ ΓΙΝΕΤΑΙ ΜΕ ΤΑΥΤΟΧΡΟΝΗ ΜΕΤΑΒΟΛΗΤΗΣ ΠΡΟΛΙΝΗΣ ΣΕ ΥΔΡΟΞΥΠΡΟΛΙΝΗ. ΤΟ ΚΟΛΛΑΓΟΝΟ ΤΥΠΟΥ IV ΤΩΝ ΝΕΟΣΧΗΜΑΤΙΖΟΜΕΝΩΝ ΒΜ ΥΠΟΛΟΓΙΣΘΗΚΕ, ΕΙΤΕ ΑΠΟ ΤΗΝ ΠΟΣΟΤΗΤΑ ΤΗΣ (U- 14C)-ΥΔΡΟΞΥΠΡΟΛΙΝΗΣ, ΕΙΤΕ ΑΠΟ ΤΗΝ ΠΟΣΟΤΗΤΑ ΤΩΝ ΚΟΛΛΑΓΟΝΟΥΧΩΝ ΠΡΟΙΟΝΤΩΝ ΑΠΟΙΚΟΔΟΜΗΣΗΣ ΜΕΤΑ ΑΠΟ ΤΗΝ ΠΕΨΗ ΜΕ ΚΛΟΣΤΡΙΔΙΚΗ ΚΟΛΛΑΓΕΝΕΣΗ. ΑΠΟ ΤΑ ΑΠΟΤΕΛΕΣΜΑΤΑ ΔΙΑΠΙΣΤΩΘΗΚΕ ΟΤΙ Ο ΜΕΓΙΣΤΟΣ ΡΥΘΜΟΣ ΤΗΣ ΒΙΟΣΥΝΘΕΣΗΣ ΤΩΝ ΚΟΛΛΑΓΟΝΟΥΧΩΝ ΠΡΩΤΕΙΝΩΝ ΤΗΣ CAM ΠΑΡΑΤΗΡΕΙΤΑΙ ΜΕΤΑΞΥ ΤΗΣ8ΗΣ ΚΑΙ ΤΗΣ 10ΗΣ ΗΜΕΡΑΣ ΑΝΑΠΤΥΞΗΣ ΤΩΝ ΕΜΒΡΥΩΝ ΚΑΙ ΟΤΙ ΑΝΤΙΣΤΟΙΧΕΙ ΣΤΗ ΦΑΣΗ ΤΗΣΡΑΓΔΑΙΑΣ ΑΓΓΕΙΟΓΕΝΕΣΗΣ. (ΠΕΡΙΚΟΠΗ)