MICROVASCULAR RESEARCH 38, 136-147 (1989) Mapping of the Microcirculation in the Chick Chorioallantoic Membrane during Normal Angiogenesis’ DAVID 0. DEFOUW, VICTOR J. RIZZO, RICHARD STEINFELD, AND RICHARD N. FEINBERG Department qf Anatomy, UMDNJ-New Jersey Medical School. 185 South Orange Avenue. Newark, New Jersey 07103-2757 Received February 14, 1989 The microcirculation within the chorioallantoic membrane (CAM) of the chick is par- ticularly 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, IO, 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 in- creased 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 microin- jections of fluorescein isothiocyanate (FITC)-dextran. were substantially less at Days IO 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. Q 1989 Academic Press. Inc. I This work was supported, in part, by a grant from the American Heart Association, New Jersey Affiliate. 136 0026~2862189 $344 Copyright Q 1989 by Academic Press, Inc. All rights of reproduction in any form reserved Printed in U.S.A.
Transcript
MICROVASCULAR RESEARCH 38, 136-147 (1989)
Mapping of the Microcirculation in the Chick Chorioallantoic Membrane during Normal Angiogenesis’
DAVID 0. DEFOUW, VICTOR J. RIZZO, RICHARD STEINFELD, AND RICHARD N. FEINBERG
Department qf Anatomy, UMDNJ-New Jersey Medical School. 185 South Orange Avenue. Newark, New Jersey 07103-2757
Received February 14, 1989
The microcirculation within the chorioallantoic membrane (CAM) of the chick is par- ticularly 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, IO, 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 in- creased 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 microin- jections of fluorescein isothiocyanate (FITC)-dextran. were substantially less at Days IO 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. Q 1989 Academic Press. Inc.
I This work was supported, in part, by a grant from the American Heart Association, New Jersey Affiliate.
136
0026~2862189 $344 Copyright Q 1989 by Academic Press, Inc. All rights of reproduction in any form reserved Printed in U.S.A.
ANGIOGENE~ IN THE CHICK (CAM) 137
INTRODUCTION
The chorioallantoic membrane (CAM) of the chick is formed by fusion of the somatic mesoderm of the chorion with the splanchnic mesoderm of the allantois during the 4th to 5th day of embryonic development. This highly vascular fusion membrane serves as the first respiratory system of the avian embryo and is solely responsible for embryonic gas exchange until Day 19 of the normal 21-day in- cubation (Rahn et al., 1974). Because of its extensive microvascular network, the CAM is a commonly used assay for studying such biological processes as gas exchange (Tazawa, 1978), angiogenesis (Barnhill and Ryan, 1983; Thompson and Brown, 1987; Folkman and Klagsbrun, 1987), and the phases of tumor vascularization and growth (Knighton et al., 1977; Folkman, 1985).
Oxygen demands of the embryo progressively increase to accommodate con- tinuous tissue differentiation and growth. Accordingly, increases of chorioallan- toic capillary volume and surface area were recorded during chick embryogenesis (Fitze-Gschwind, 1973). In addition, total length of the CAM vessels was highly correlated with overall size of the CAM (Tanaka et al., 1986). The diffusion capacity of oxygen across the CAM was reported to increase continuously during embryonic incubation (Tazawa, 1978). This increase is likely the result, in part, of both an expansion of the capillary network and location of the capillaries within the CAM. Initially, the capillaries reside in the mesoderm immediately beneath the chorionic ectoderm (Gonate et al., 1964) and by Day 14 the capillaries penetrate into the chorionic epithelium and either remain separated from the shell membrane by attenuated chorionic epithelial cells (Narbaitz, 1977) or make direct contact with the shell membrane (Fuchs and Lindenbaum, 1988). Further, in a previous study from this laboratory, ultrastructural characteristics of these capillaries were observed to vary as a result of progressive endothelial cytodif- ferentiation during embryogenesis (Shumko et al., 1988).
Although chronological expansion of the CAM capillary network is well es- tablished, descriptions of the branching patterns of CAM pre- and postcapillary microvessels during embryonic maturation have not been recorded. Since pre- and postcapillary microvessels were observed to be involved in microvascular remodeling that is associated with postnatal tissue maturation (Sarelius et al., 1981; Unthank and Bohlen, 1987), the present study provides a morphometric description of the remodeling of precapillary, capillary, and postcapillary seg- ments of the CAM during chick embryonic development. The results of this study present a quantitative description of the CAM microcirculation and thus provide a structural basis for the CAM angiogenic or gas exchange assay and for additional functional studies during microvascular histodifferentiation.
METHODS
White Leghorn chicken embryos were incubated in an humidified environment at 37” for 3 days in OVO. The shells were then cracked open aseptically and the embryonated eggs were placed in plastic culture dishes according to an estab- lished shell-less culture technique (Auerbach et al., 1974). The embryos were then incubated in a humidified environment at 37”. This culture technique enabled unobstructed viewing of the CAM microcirculation during its functional lifespan.
138 DEFOUW ET AL.
On the basis of our previous observations (Shumko et al., 1988), three observation periods were selected: Days 6, 10, and 14 of incubation.
For microscopic evaluation, the culture dishes were removed from the incu- bator and placed on a temperature-controlled microscope stage (Midland Ross). The pre- and postcapillary microvessels were then observed with a Nikon SMZ- 10 stereomicroscope. The linear dimensions of the CAM were recorded and, prior to microscopic observation, a planar area of 0.49 cm2 (0.7 x 0.7 cm) was demarcated on the surface of the CAM (Fig. 1). Given observed blood flow patterns, the pre- and postcapillary microvessels were identified within the planar area and descriptively recorded. This area was then photographed at 30 x final magnification using Kodak Tri X (ASA 400) black and white film. Two photo- micrographs were required to cover the entire area and a single photomontage was made for morphometric analyses. At Day 6, a single planar area provided a sufficient sample of the CAM; however, at Days IO and 14 two planar areas were sampled to adequately record the CAM microvessels. A total of 20 pho- tomicrographs was recorded for each of the three observation periods.
Morphometric analyses of the micrographs were performed with the aid of a Zeiss MOP image analyzer. Initially, the centripetal ordering method of micro- vascular mapping (Fenton and Zweifach, 1981) was applied to the pre- and postcapillary microvessels. This method of ordering sets the capillaries as the
FIG. 1. A single planar area is depicted on the surface of a 14-day CAM. A fifth-order precapillary vessel (large arrowhead) and a fourth-order postcapillary vessel (small arrowhead) are identified in an attempt to categorize the smaller order microvessels (see Fig. 2) which serve as the principal focus of this study. 9x.
ANGIOGENESI~ IN THE CHICK (CAM) 139
point of reference and the most distal precapillary vessel or the most proximal postcapillary vessel was assigned Order 1. When two first-order vessels con- verged an Order 2 vessel was formed. Likewise, the convergence of two second- order vessels formed an Order 3 vessel. In the case of the convergence of two vessels of unequal order, the highest order was retained. In both the pre- and postcapillary networks three orders of vessels were analyzed.
After the number of branches in each order was counted on the photomontages, diameters and lengths of the branches in each order were measured and mean values were calculated. Statistical comparisons of the mean values were per- formed using one-way analysis of variance and Duncan’s multiple comparison procedure (Duncan, 1955), and P < 0.05 was accepted as significant.
In a separate series the CAM capillaries, as presented in the shell-less culture preparations, were viewed with an Olympus BHA microscope for epi-illumination of microinjected fluorescein isothiocyanate (FITC)-dextran 150 (Ley and Arfors, 1986). A 100-W mercury DC lamp source along with a heat-absorbing filter and the appropriate exciter (490 nm) and barrier (515 nm) filters served to illuminate the tagged macromolecules within the capillaries. Such illumination served to clearly depict the capillary networks, which were not distinctly visualized with bright field microscopy. Microinjections of FITC-dextran (5% in avian Ringer’s solution) were performed with a Pica-Injector PLI-100 (Medical Systems, Inc.). Initially, glass capillary tubes were pulled on a vertical pipet puller (David Kopf), beveled to a tip size of 5-10 pm, and siliconized. The micropipets were then inserted into a micromanipulator (Narishige MN-151), which was mounted on the microscope stage. A Dage video camera and video monitor, in conjunction with the micromanipulator, enabled insertion of the micropipets into a second- order precapillary vessel. A dual viewing body in the observation tube of the microscope served to provide 35-mm photographic records of the CAM capillary networks during the microinjections. Kodak T-max P3200 black and white film was used to compensate for the low light intensities of the microscopic fields and for inopportune motion created by intermittent movement of the embryo, particularly at Days 10 and 14 of development. To minimize the deleterious effects of blue light irradiation of FITC-dextran (Bekker et al., 1987), the mi- croinjections were limited to 30-set durations. Using the pica-injector, injectates were delivered at a rate of 0.03 mm/set which presented approximately 0.2 ~1 FITC-dextran into the localized capillary network during each 30-set injection period.
Fifteen to twenty fluorescent photomicrographs at 3 10 x final magnification, obtained from several specimens in each of the three observation periods, were used to record measurements of intercapillary distances. Mean values were then calculated and compared statistically as described above.
RESULTS
After 6 days of incubation, the CAM presented an average surface area of approximately 6 cm’. By Day 10, and extending to Day 14, mean surface area of the CAM was approximately 65 cm’. This expansion of CAM surface area was accompanied by an increased complexity of the patterns of pre- and post- capillary microvessels. The numbers of first-, and second-, and third-order vessels
140 DEFOUW ET AL.
TABLE 1 NUMBERS OF PRE- AND POSTCAPILLARY VESSELS PER SQUARE CENTIMETER CAM AND MEAN
INTERCAPILLARY DISTANCES
Vessel order
Precapillary 1 2 3
Postcapillary 1 2 3
Intercapillary distance (wd
Day 6 Day IO
94 2 2Y 167 + 41d 33 t gd 57 k 12d 12 -t ed 16 +- P’
39 _f 12 98 k 33 145 5 33 t 12 6? 2 102 4
25+ 4’ 152 2
Day 14
249 t Fib 80 t 17h 215 8’
214 k 83” 76 2 27h 17 2 IO 16+ 2
’ Mean values k one standard deviation. ’ Day 14 > Day 10 > Day 6. ’ Order 1 > Order 2 > Order 3. ’ Precapillary number > postcapillary number in respective orders. ’ Day 6 > Days 10 and 14.
are presented in Table 1. For both the pre- and postcapillary compartments the number of first-order vessels exceeded the number of second-order vessels while second-order vessels outnumbered third-order vessels during each observation period. In addition, first- and second-order vessels progressively increased in number from Day 6 to Day 14. Third-order vessels, however, remained essentially constant in number during the observed periods of incubation. Interestingly, the number of precapillary vessels was greater than postcapillary vessels in their respective orders at Days 6 and 10; however, by Day 14 this difference was no longer significant.
The most common arrangement (62% of the cases) of both pre- and postcap- illary vessels was presented by two first-order vessels joining to form a second- order vessel (Fig. 2). In 11% of the cases, one or more additional first-order vessels joined the second-order vessel. In addition, a single first-order vessel joined either a second-order vessel (20% of the cases) or a third-order vessel in 7% of the cases. This pattern of pre- and postcapillary vessel branching was observed continuously from Day 6 to Day 14 of incubation. The intercapillary distances (Fig. 3), on the other hand, were not uniform thoughout the incubation. As presented in Table 1, dimensions of the mesodermal interstitium between capillaries at Day 6 were substantially greater than those recorded at Days IO and 14. Further, the intercapillary distances were similar at Days 10 and 14.
Mean lengths of the first-, second-, and third-order microvessels are presented in Table 2. For each pre- and postcapillary vessel order, average length was inversely related to vessel number during all observation periods. Thus, mean lengths of the third-order vessels were significantly greater than those of second- order vessels, which, in turn, were greater than those of the first-order vessels. The first-, second-, and third-order vessels of both the pre- and postcapillary compartments displayed a slight decrease in average length between Days 6 and 10. By Day 14, mean lengths of each of the three vessel orders were significantly reduced relative to the original measurements recorded at Day 6.
ANGIOGENESIS IN THE CHICK (CAM) 141
FIG. 2. This field serves to illustrate the center of the planar area depicted in Fig. I. In the lower right, a portion of the precapillary compartment is labeled according to the centripetal ordering method used in this study. Likewise, a postcapillary bed is labeled in the upper left. Note the order- one, two, and three microvessels in each compartment. 30x.
Average diameters of the pre- and postcapillary microvessels are presented in Table 3. Diameters of the first-, second-, and third-order vessels were unchanged during the observed periods of incubation. As was the case for microvessel lengths, average diameters of third-order vessels were greater than those of the second-order vessels which, in turn, were greater than mean diameters of the first-order vessels. Further, mean vessel diameters, like average vessel lengths, were comparable in the three vessel orders of the pre- and postcapillary com- partments, respectively.
DISCUSSION
In the shell-less culture of chicken embryos the CAM microcirculation is readily available for microscopic observation. The surface area measurements of the CAM obtained presently are similar to those reported previously in ovu (Fitze- Gschwind, 1973). Thus, the culture technique affords an unobstructed two-di- mensional view of the entire CAM microcirculation without distorting normal CAM dimensions during its histodifferentiation. Whether the shell-less culture preparations accurately reflect all events occurring during embryogenesis in ova, however, remains to be verified.
Based on the data presented in Table I, the average branching ratio of daughter to parent vessels (both pre- and postcapillary) was 3.14. This value is similar to that reported previously in skeletal muscle of adult rats (Koller and Johnson, 1986). The fact that the branching ratio remained essentially constant from Day
142 DEFOUW ET AL.
FIG. (c, 179 Days 1
frequel
3. Intercapillary distances are compared between Days 6 (a, 175 x ). 10 (b, 179x), and 14 x ) of incubation. At Day 6 the distance between capillaries is greater than that observed at 0 or 14. Greater image clarity at Day 6 serves to depict erythrocytes intermixed with FITC- n within the capillaries. Such clarity was not obtained at Days 10 and 14 because of more it motion created by the developing embryo.
ANGIOGENE~ IN THE CHICK (CAM) 143
TABLE 2 MEAN LENGTHS (mm) OF PRE- AND POSTCAPILLARY CAM VESSELS~
y Mean values + one standard deviation. h Order 3 > Order 2 > Order 1. ’ Day 14 < Day 6.
6 to Day 14 of incubation is consistent with the interpretation that angiogenesis (expressed as the progressive increase in number of microvessels) is programmed to create and to maintain uniform patterns of microvessel topography. In an ideal bifurcating network with a branching ratio of 3.0, the frequency of a first- order vessel joining another first-order vessel would be 0.67. In the present study this was observed to occur in 62% of the cases. Further, in such an ideal network, the frequency of a first-order vessel joining a second-order vessel would be 0.33, while the frequency of it joining a third-order vessel would be zero. However, in the CAM microvascular network (both pre- and postcapillary compartments), first-order vessels joined a second-order vessel in 20% of the cases and a third- order vessel in 7% of the cases.
The CAM microcirculation is supplied by two primary chorioallantoic arteries and drained by a single chorioallantoic vein. These primary vessels are several vessel orders removed from the third-order pre- and postcapillary vessels de- scribed in this study. From Day 6 to Day 14, the third-order microvessels did not increase significantly in number, while the numbers of first- and second- order vessels were substantially increased. This observation is consistent with the concept that angiogenesis within the CAM was the result, in part, of con- tinuous neoformation of first- and second-order microvessels during embryo- genesis.
Previous studies provided evidence consistent with the hypothesis that pre- capillary microvessels provide a primary site of oxygen exchange in adult skeletal
TABLE 3 MEAN DIAMETERS (pm) OF PRE- AND POSTCAPILLARY CAM VESSELS”
Vessel order Day 6
Precapillary 1 39-r- 7 2 65 k 15 3 94 t 19h
Postcapillary 1 46-c 7 2 81 2 14 3 136 2 31”
u Mean values ? one standard deviation. ’ Order 3 > Order 2 > Order 1.
Day 10 Day 14
45 t II 47+ 6 71 ? 12 78 k II
112 ? 27” 116 f 23” 54 k 10 522 8 76 -c 14 75 t 12
113 k 17h 106 k lSh
144 DEFOUW ET AL.
muscle (Pope1 and Gross, 1979; Pittman, 1987). Since the CAM serves as the principal respiratory system of the avian embryo, additional evidence is required to determine the role of the continuously expanding precapillary (or postcapillary) vascular network in gaseous exchange during embryonic development.
The period of incubation extending from Day 6 to Day 10 was characterized by a more rapid expansion of precapillary microvessels than their postcapillary counterparts. Ausprunk et al. (1974) previously reported intense incorporation of tritiated thymidine by CAM capillary endothelial cells from Days 8 to 10 of incubation. Labeling of closely associated pre- and postcapillary endothelial cells was, however, considerably less frequent. This occurrence of capillary endo- thelial cell proliferation concomitant with neoformation of first- and second-order precapillary (and to lesser extent postcapillary) microvessels provides support to the hypothesis that capillaries served as the source of first- and second-order microvascular endothelia during Days 6 to 10 of incubation. The fact that the number of third-order microvessels remained constant during this period is, on the other hand, consistent with the alternate interpretation that possible prolif- eration or elongation of third-order vessel endothelia served as the source for the progressive increase in numbers of first- and second-order microvessels. Whether the unequal number of principal supplying (arterial) and draining (ve- nous) vessels of the CAM contributes to the presence of greater numbers of third-order precapillary vessels than their postcapillary counterparts up to Day 10 also remains an intriguing question. Although it is generally accepted that new capillary formation, in response to a variety of angiogenic stimuli, originates from small venules or preexisting capillaries (Folkman, 1983, the source re- sponsible for pre- or postcapillary microvessel neoformation during CAM an- giogenesis, as described above, remains uncertain. Interestingly, Thompson and Brown (1987) reported that the application of histamine to the CAM at Day 10 was associated with an increase in number of distal arterial branches while capillary density remained unchanged. The source of these newly formed pre- capillary vessels was, however, not determined.
According to Ausprunk et ul. (1974), capillary angiogenesis is completed by Day 11 in the normal CAM. The present measurements of intercapillary distances are also consistent with cessation of capillary angiogenesis after Day 10 of in- cubation. Between Days 6 and 10, intercapillary distances were substantially reduced; however, capillary density, as measured by intercapillary distances, remained constant between Days 10 and 14. Thus, expansion of the capillary networks appears to cease between Days 10 and 14 of incubation. If it is assumed that capillary endothelial cell proliferation does not occur from Days 10 to 14, it would seem unlikely that newly formed capillary endothelial cells contributed to the marked proliferation of first- and second-order pre- and postcapillary microvessels during this period of incubation. Elongation of existing capillary endothelial cells, on the other hand, might contribute to the neoformation of first- and second-order microvessels during this period.
The fact that average lengths of the first-, second-, and third-order microvessels were significantly reduced by Day 14 is consonant with the interpretation that consecutive branching of the respective vessel orders might serve to increase the total number of vessels while simultaneously decreasing the length of each microvessel within the expanding network. The concept that preexisting capil-
ANGIOGENESIS IN THE CHICK (CAM) 145
laries at Day 10 undergo histodifferentiation to become pre- or postcapillary microvessels by Day 14 also requires further evaluation. Clearly, an accurate description of the pattern of angiogenesis within the CAM awaits additional experimental evidence.
Between Days 10 and 14, the number of postcapillary microvessels increased at a faster rate than their precapillary counterparts. Since the number of pre- capillary vessels exceeded the number of postcapillary vessels up to Day 10, the unequal rates of angiogenesis between Days 10 and 14 created similar numbers of pre- and postcapillary vessels by Day 14. The mechanisms responsible for unequal proliferation rates of pre- and postcapillary vessels during embryogenesis remain uncertain.
Although, the number of precapillary vessels was greater than the number of postcapillary vessels up to Day 10, mean diameters and lengths of the postcap- illary vessels tended to be greater than those recorded for the precapillary vessels. Given this data, the vascular volume/cm’ CAM presented at Day 10 by the first three orders of precapillary vessels was estimated at approximately 5 x lop4 cm3 while that of the postcapillary vessels was estimated at 4.25 x 10e4 cm3. Thus, up to Day 10, the respective variations in microvessel dimensions appar- ently compensated for the greater relative frequency of precapillary vessels; hence, pre- and postcapillary microvascular volumes were essentially comparable at this time. By Day 14, the estimates of pre- and postcapillary volumes/cm2 CAM increased to approximately 7 x 10e4 cm3 and 5.5 x 1O-4 cm3, respectively. Thus, at Day 14, blood volume within the first three orders of microvessels in the pre- and postcapillary compartments was approximately 40 ~1 in each com- partment. Since the present microscopic observations served to identify only blood-perfused microvessels, the presence of unperfused vessels, which might create a microvascular reserve capacity, cannot be denied; however, their ex- istence was not evaluated in this study.
Previous studies presented evidence of fivefold increases in both systolic and diastolic pressures in CAM arteries from Day 6 to Day 14 of incubation (VanMierop and Bertuch, 1967; Girard, 1973). The fact that progressive elevation of vascular hydrostatic pressure contributed to the present observation of in- creased numbers of first- and second-order CAM microvessels is consistent with the general concept that blood pressure represents an important influence on microvascular histodifferentiation (Gonzales-Crusci, 1971; Guard, 1973). Since matrix molecules (e.g.. collagen and glycosaminoglycans) were also correlated with morphological events of CAM angiogenesis (Ausprunk, 1986), identification of the factors responsible for neoformation of CAM microvessels during em- bryogenesis remains an important question.
In summary, the results of this study provide a structural framework, based on quantitative descriptions, of the CAM microcirculation. This framework serves as a basis for the CAM angiogenic assay and for additional functional studies during microvascular histodifferentiation. Further, several hypotheses were presented regarding the precise endothelial precursor for progressive ex- pansion of the first- and second-order pre- and postcapillary microvessels during CAM angiogenesis. Additional experimental evidence is required to ascertain the mechanisms of microvascular modeling that regulate the spatial and temporal development of this embryonic network.
146 DEFOUW ET AL.
ACKNOWLEDGMENTS
The authors thank Ms. Lisa Sweetman for her technical assistance and Ms. Marge Green for typing of the manuscript.
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