Victor Rizzo

Mapping of the microcirculation in the chick chorioallantoic membrane during normal angiogenesis.

The microcirculation within the chorioallantoic membrane (CAM) of the chick is particularly well suited for in vivo observation and has been used extensively as an assay to detect angiogenic activity. Although progressive chronological expansion of the CAM capillary network occurs normally during embryogenesis, descriptions of the branching patterns of CAM pre- and postcapillary microvessels during embryonic development have not been recorded. In the present study chick embryos were incubated, using an established shell-less culture technique, and observed in vivo at Days 6, 10, and 14 of embryonic development. Morphometric analyses of photomicrographs of CAM microvessels were based upon the centripetal ordering method of microvascular mapping of the first three orders of pre- and postcapillary microvessels with the capillaries serving as the initial point of reference. For both pre- and postcapillary vessels, the number of first-order vessels exceeded the number of second-order vessels which, in turn, outnumbered third-order vessels during each observation period. First- and second-order vessels progressively increased in number from Day 6 to Day 14; however, the number of third-order vessels remained essentially constant during this period. Further, the number of precapillary vessels was greater than postcapillary vessels in their respective orders at Days 6 and 10; however, by Day 14 the numbers were comparable. Average diameters and lengths of the third-order vessels were greater than the second-order vessels which, in turn, were greater than the first-order vessels in both the pre- and postcapillary compartments. Further, mean lengths of each of the three vessel orders in both compartments decreased progressively and by Day 14 were significantly less than at Day 6. Average diameters of each vessel order, on the other hand, remained unchanged from Day 6 to Day 14. Finally, intercapillary distances, based on measurements from fluorescent micrographs obtained after microinjections of fluorescein isothiocyanate (FITC)-dextran, were substantially less at Day 10 and 14 than at Day 6. Based on these morphometric data, the endothelial precursor responsible for continuous neoformation of first- and second-order microvessels during embryogenesis remains uncertain. Whether existing first-, second-, or third-order vessel endothelia serve as this precursor or histodifferentiation of existing capillaries enables continuous expansion of the first- and second-order microvessels remains to be tested.

Macromolecular selectivity of chick chorioallantoic membrane microvessels during normal angiogenesis and endothelial differentiation

Progressive angiogenesis and endothelial differentiation in the chick chorioallantoic membrane (CAM) serve to accommodate oxygen demands of the growing embryo. The present study evaluated CAM microvascular endothelial permselectivity during the most rapid phase of angiogenesis (day 10) and after initiation of endothelial cytodifferentiation (day 14). Chick embryos were incubated using established shell-less culture techniques for intravital and ultrastructural observations. Systemic microinjections of FITC-dextrans (40, 70 and 150 KDa) provided an index of endothelial permselectivity after 2.5 min and 10 min perfusions. Ultrastructural examinations of the same dextran probes served to detect small, intermittent foci within the perivascular interstitium. Although minor variations of dextran particle distributions around specific segments of the microcirculation were observed ultrastructurally, perivascular accumulation was not sufficient to elicit a detectable fluorescent signal. Thus, substantial accumulation of the graded-dextran series in the perivascular interstitium was not detected. Morphometric analyses of the precapillary, capillary, and postcapillary microvascular segments served to demonstrate a continuous endothelium which displayed cytoplasmic attenuation at day 14. Plasmalemmal vesicles were few and uniform within the microvascular units at day 10. A three-fold increase in vesicle densities characterized the precapillary endothelia at day 14. Average widths of the endothelial junctional clefts were homogeneous within the segmental microvascular endothelia at both days 10 and 14. Junctional cleft lengths were also homogeneous, except the significantly longer capillary endothelial clefts observed at day 10. These results are consistent with the concept that, despite certain differences in segmental vesicle densities and junctional cleft lengths, neovascularization of the CAM is achieved without excessive macromolecular efflux across the microvascular endothelia.

Differentiation of the microvascular endothelium during early angiogenesis and respiratory onset in the chick chorioallantoic membrane

The present study served to determine the extent of microvascular endothelial differentiation during early stages of morphogenesis (days 4.5-5.5 of the 21-day incubation) in the chick chorioallantoic membrane (CAM). CAMs, which serve as the embryonic lung, were prepared for intravital injections of a graded series of FITC-dextrans and subsequent ultrastructural morphometric analyses of the microvascular units. The precapillary, capillary, and postcapillary microvascular segments presented a continuous endothelium that was substantially thicker than that of adult lung endothelia (DeFouw, 1988). Further, plasmalemmal vesicles were uniformly sparse, while endothelial vacuoles, of variable diameters, were present continuously in the proliferating microvascular units. Average widths and depths of the interendothelial clefts were uniform and suggested complete structural differentiation from the onset of CAM morphogenesis. Based on our recent estimates of CAM microvascular permeability coefficients (Rizzo et al., 1995), the observed endothelial ultrastructure was associated with microvascular selectivity comparable to that of adult pulmonary microvessels (Lanken et al., 1985). Therefore, despite incomplete ultrastructural differentiation of the early CAM microvascular endothelium, these angiogenic microvessels presented adult-like barrier properties. Further they were less permeable than (Wu et al., 1993; Yuan et al., 1993) and ultrastructurally distinct from (Kohn et al., 1992) certain tumorigenic microvessels. Thus, angiogenesis is likely not a routinely homogeneous process, and CAM microvascular permeability characteristics may be teleologically significant.

MICROVESSELS OF THE CHICK CHORIOALLANTOIC MEMBRANE UNIFORMLY RESTRICT ALBUMIN EXTRAVASATION DURING ANGIOGENESIS AND ENDOTHELIAL CYTODIFFERENTIATION

The present study served to evaluate extravasation of albumin across the microvascular endothelium of the chick chorioallantoic membrane (CAM) during the establishment of endothelial barrier functions (days 4.5 to 6.0), during a rapid phase of normigenesis (day 10), and after initiation of endothelial cytodifferentiation (day 14). CAM preparations in shell-less cultures were evaluated by intravital fluorescence confocal microscopy and videodensitometry after microinjections of chicken serum albumin (CSA) conjugated to Fluorescein isothiocyanate (FITC) (1% in Avian Ringers). At each observed temporal stage, FITC-CSA extravasation from the CAM pre-capillaries, capillaries, and post-capillaries was uniformly negligible. Since the endothelial glycocalyx imparts a net negative luminal charge, impedance of CSA transport by mutual charge repulsion was also evaluated. Accordingly, CAM microvessels were pre-infused with protamine sulfate (100 microliters/ml) to neutralize the negative charge. However, interstitial accumulation of FITC-CSA remained negligible. Thus, the presence of tight junctional clefts and paucity of plasmalemmal vesicles during CAM normigenesis probably serve as the principal morphologic correlates to the observed albumin restriction. Further, the normigenic CAM endothelium presents a more restrictive barrier to macromolecules than that of most angiogenic endothelia.

Microvascular Permselectivity in the Chick Chorioallantoic Membrane during Endothelial Cell Senescence

The chorioallantoic membrane (CAM) of the chick embryo represents an in vivo model to evaluate microvascular function during the sequential phases of endothelial proliferation (angiogenesis), cytodifferentiation, and senescence. The principal focus of this study was to characterize microvascular barrier functions of the CAM endothelium during its nonproliferating, aging phase (senescence). A graded series of fluorescein isothiocyanate (FITC) dextrans served to index macromolecular selectivity of the senescent capillary endothelium. Extravasation of FITC dextrans 40 and 150 was restricted, while FITC dextran 10 progressively accumulated within the intercapillary interstitia during 15-min perfusion periods. Endothelial vesicle densities were greater in the first- and second-order pre- and first- order postcapillary endothelia (28 +/- 8/micron2) than those recorded for the capillary endothelium (8 +/- 3/micron2). Junctional cleft lengths (luminal to abluminal distances), on the other hand, were greater in the capillary endothelium (1.08 +/- 0.50 microns) than those recorded for the second-order pre- and postcapillaries (0.46 +/- 0.03 micron). Junctional cleft widths were segmentally uniform (20 nm) in the microvascular units. That permselectivity of the segmental microvascular endothelia was homogeneous is ultrastructurally consistent with the uniform junctional cleft widths rather than the heterogeneous cleft lengths and vesicle densities. The CAM serves as the principal respiratory exchange surface of the embryo. Thus, maintenance of colloid oncotic balance across the microvascular endothelium is likely critical to gaseous exchange. The retention of a selective barrier during the senescent phase of the CAM endothelium serves to support this concept.

Degranulation of Mast Cells in the Chick Chorioallantoic Membrane Does Not Increase Macromolecular Extravasation During Normal Angiogenesis

Objective: To test the hypothesis that stimulation of mast cell degranulation during normal angiogenesis in the chick chorioallantoic membrane (CAM) serves to acutely activate macromolecular transendothelial pathways.