David Defouw - Academia.edu (original) (raw)
Papers by David Defouw
The Faseb Journal, Apr 1, 2010
This study examined the effects of acute administration of bleomycin (BLM) on lung liquid and pro... more This study examined the effects of acute administration of bleomycin (BLM) on lung liquid and protein exchange in anesthetized sheep prepared with caudal mediastinal lung lymph fistulas. Six sheep received BLM (15 U IV) after a baseline period, while seven others were given BLM after a steady state was obtained following elevation of left atrial pressure (PLA), a procedure intended to minimize changes in pulmonary microvascular surface area and produce high lung lymph flow (QL). Plasma and lung lymph angiotensin converting enzyme (ACE) activities were measured in order to independently assess the effects of BLM on pulmonary endothelial integrity. QL rose significantly in all animals following BLM. The ratio of lymph to plasma protein concentration (CL/CP) did not change in the group given BLM alone, and fell continuously during the period of PLA elevation after BLM in that group. Plasma and lung lymph ACE activities were unchanged following BLM administration in either group. The ultrastructure of the gas-exchanging region of lungs from animals in each group was examined by transmission electron microscopy. The data suggest that acute administration of a low dose of BLM does not increase pulmonary microvascular permeability, but may induce an increase in perfused pulmonary microvascular surface area responsible for increased QL.
Journal of Vascular Research, 1993
Cultured canine jugular vein endothelial cells were seeded on polycarbonate filters to create an ... more Cultured canine jugular vein endothelial cells were seeded on polycarbonate filters to create an in vitro permeability assay. The calculated diffusive permeability coefficient for FITC-BSA across untreated monolayers was 1.1 +/- 0.4 x 10(-6) cm/s. After 15-min incubations with either histamine or bradykinin, the resistance to albumin flux across the monolayers was reduced significantly. Diffusive albumin permeability coefficients were 3.4 +/- 1.8 x 10(-6) and 4.1 +/- 2.0 x 10(-6) cm/s, respectively. Ultrastructural morphometric analyses of the endothelial cell monolayers served to define uniform dimensions of intercellular clefts and similar plasmalemmal vesicle densities in the untreated and the vasomediator-activated monolayers. These results are consistent with the interpretation that the vasomediator-activated pathway across the venous endothelial monolayers is not dependent on sustained intercellular gap formation or sustained expansion of the plasmalemmal vesicle population for the 15-min observation periods. Whether the increased albumin flux is dependent on transient gap formation or on physical changes within the venous endothelial cell glycocalyx remains to be tested.
Objective: To test the hypothesis that stimulation of mast cell deganulation during normal angiog... more Objective: To test the hypothesis that stimulation of mast cell deganulation during normal angiogenesis in the chick chorioallantoic membrane (CAM) serves to acutely activate macromolecular transendothelial pathways.
Microvascular Research, Mar 31, 1997
The American review of respiratory disease
The Anatomical record, 1988
This study presents a quantitative description of ultrastructural changes associated with focal h... more This study presents a quantitative description of ultrastructural changes associated with focal histodifferentiation of the chick chorioallantoic membrane (CAM) microvasculature. Early vessels (4-8 days of a 21-day incubation period) were lined by a thin, but continuous endothelium which was characterized by punctate appositional contacts, an incomplete or absent basal lamina, and few plasmalemmal vesicles. During this stage, there was little ultrastructural distinction between CAM arterioles, capillaries, and venules. During the intermediate stage (9-13 days), the arteriolar endothelium displayed more extensive junctional apposition. In addition, the interstitial (periendothelial) space contained more collagen fibrils and increased cell volume densities, some of which resembled presumptive smooth muscle cells. In contrast to the arterioles, endothelial appositions of the venules and capillaries remained punctate during the intermediate stage. The interstitial space of the venules d...
The Anatomical Record, 1995
The chorioallantoic membrane (CAM) of the chick embryo expands during embryogenesis to meet the i... more The chorioallantoic membrane (CAM) of the chick embryo expands during embryogenesis to meet the increased oxygen demands during growth and differentiation. Temporal and spatial glycosylation patterns of CAM ectodermal and endodermal proteins likely contribute to differentiation of the functional attributes of the CAM. Using lectins for light and electron microscopic observations, we studied the patterns of glycoconjugate expression on the ectoderm and endoderm of the chorioallantoic membrane (CAM) of the chick at days 4.5, 5.0, 5.5, 6.0, and 10 of morphogenesis. For light microscopy, samples of unfixed CAM were incubated with the following FITC lectins: Con A, DBA, GSA-I, GSA-II, PNA, SBA, UEA-I, and WGA. All lectins, except GSA-I and -II, gave positive results. The positive lectins, labeled with HRP, served to ultrastructurally localize PNA, SBA, and WGA, but not DBA binding to the luminal surface of the endoderm. UEA-I and Con A bound similarly except on day 10 when UEA-I no longer bound. On the ectodermal surface, only WGA bound at all times studied. PNA and SBA binding were present from days 5.0 to 6.0 but absent at days 4.5 and 10. DBA binding occurred through day 5.0 but was absent thereafter. UEA-I bound to the ectoderm at days 4.5, 5.0, and 10 but not days 5.5 and 6.0. Con A bound only on days 5.0 and 10. That the ultrastructurally similar ectoderm and endoderm of the CAM display functional differences conforms to the hypothesis that differential expression of glycoconjugate microdomains likely contributes to such functional specialization.
Annals of Vascular Surgery, 1992
Repair of occult arterial injuries is advocated to prevent thrombosis, arteriovenous fistula, and... more Repair of occult arterial injuries is advocated to prevent thrombosis, arteriovenous fistula, and pseudoaneurysm formation. However, recent clinical series describe the healing of arterial intimal injuries and recommend nonoperative therapy. To investigate the arterial wall response to intimal injury, we created intimal flaps in 46 canine femoral arteries. The intimal flaps were imaged by arteriography, angioscopy, and intravascular ultrasound acutely, and at one and three weeks and five months post-injury. Lumen area was measured using caliper techniques (arteriography) and computerized video planimetry (angioscopy, intravascular ultrasound). Intimal and medial thickness were measured by intravascular ultrasound prior to harvest for histologic evaluation by light microscopy. Analysis of 32 patent arteries was performed after exclusion of 14 thrombosed arteries. Residual lumen area (mm 2) correlated closely among the imaging modalities at one week (8.7 +-1.1, 7.3 _ 2.0, 6.9 +-1.8), three weeks (4.2 +_ 0.9, 2.9 +_ 1.0, 2.7 _ 0.8), and five months (5.3 -+ 0.9, 5.0 -0.5, 5.0 -+ 0.9). Maximal intimal and medial thickness occurred three weeks post-injury, coincident with the maximal reduction in lumen area. Although intimal injuries can cause acute and delayed arterial thromboses, observation may be appropriate in selected cases. The evaluation of those patients chosen for nonoperative therapy should extend beyond three weeks, as this is the time of maximal arterial wall response with a continued potential for adverse clinical events. (Ann Vasc Surg 1992;6:50-54).
Respiration Physiology, 1993
We examined the effects of acute expansion of extracellular fluid volume (ECFV) on lung fluid bal... more We examined the effects of acute expansion of extracellular fluid volume (ECFV) on lung fluid balance and the ultrastructure of the pulmonary air-blood barrier in chickens (Gallus domesticus). We compared changes in extravascular lung water content (EVLW) to the sum of mean pulmonary capillary blood pressure and plasma protein osmotic pressure (nc), as a measure of net intravascular filtration pressure (NIFP), produced by graded infusion of avian Ringer's solution. NIFP increased with each volume load largely as a result of decreased nc resulting in progressive increase in EVLW. Progressive interstitial edema occurred with fluid accumulation restricted to the inter air capillary septa, sparing the gas exchanging regions of the air-blood barrier. This was associated with increased thickness of the septa and increased pulmonary capillary endothelial vesiculation. The effect of increased ECFV on pulmonary hemodynamics and lung fluid balance in Gallus is similar to that in mammals.
Microvascular Research, 1996
Using shell-less cultures of chick embryos, the effect of mast cell degranulation on normal rates... more Using shell-less cultures of chick embryos, the effect of mast cell degranulation on normal rates of angiogenesis in the chorioallantoic membrane (CAM) was recorded from Day 6 to Day 9. Based on the centripetal ordering method with capillaries as the initial point of reference, the first two orders of pre-and postcapillary microvessels were evaluated. After infusion of compound 48/80 into the allantoic cavity, the normal proliferation rate of order-1 pre-and postcapillaries was markedly increased. Increases of second-order vessel numerical densities (number/cm 2 ) were less than those of the order-1 vessels. Thus, length densities (mean length 1 numerical density) were increased for the order-1 vessels only. During the accelerated rate of CAM angiogenesis, endothelial restriction of FITC-dextran 150 remained uniformly high. Hence, secreted mast cell products likely worked synergistically with endogenous CAM angiogenic factors between Days 6 and 9, without increasing rates of macromolecular extravasation. However, histamine injection into the allantoic cavity at Day 10, in conjunction with calcium supplementation of the shell-less cultures at Day 9, elevated the rate of FITC-dextran 150 extravasation. Whether differentiation of this transendothelial pathway is required for enhancement of CAM angiogenesis after Day 10 remains uncertain. ᭧
The Anatomical Record, 1996
Background: Intraalveolar bubbles and bubble films have been shown to be part of the normal alveo... more Background: Intraalveolar bubbles and bubble films have been shown to be part of the normal alveolar architecture in vivo from birth through the first 2 days of extrauterine life of rabbit pups (Scarpelli et al., 1996a. Anat. Rec. 244:344-357). The intraluminal boundary between airway free gas and alveolar bubbles at the level of respiratory bronchioles is established within 1 hour after birth. We now examine the lung through the rest of development, namely, 2 weeks, 1,2, and 3 months, and adulthood.
The Anatomical Record, 1997
Background: Having confirmed (Scarpelli et al. 1996. Anat. Rec. 244:344-357 and 246:245-270) the ... more Background: Having confirmed (Scarpelli et al. 1996. Anat. Rec. 244:344-357 and 246:245-270) the discovery of intraalveolar bubbles and films as the normal anatomical infrastructure of aerated alveoli at all ages, we now address three questions. Why have these structures been so elusive? Visible in fresh lungs from the in vivo state, can they be preserved by known laboratory methods? Can they be preserved intact for study in tissue sections?
Tissue and Cell, 1995
The present study served to determine the extent of microvascular endothelial differentiation dur... more 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). CAM's, 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.
Tissue and Cell, 1996
It is generally accepted that luminal surfaces of adult microvascular endothelia present an anion... more It is generally accepted that luminal surfaces of adult microvascular endothelia present an anionic barrier that limits passage of anionic macromolecules. To assess the ontogeny of the barrier, temporal and spatial expression of endothelial anionic sites was evaluated in the chorioallantoic membrane of chicken embryos from days 4.5 to 18 of incubation. After an initial flush, the vessels were perfused with cationic ferritin (CF, 1.0 mg/ml in PBS) for 2 min. Following a second flush to remove unbound CF, the chick chorioal-Iontoic membranes (CAMs) were fixed and processed for electron microscopy. Continuous CF binding was revealed on the luminal endothelium, the junctional clefts and the plasmalemmal vesicles from days 4.5 to 14. However, by day 18, anionic sites had become discontinuous. Prior perfusion with protamine sulfate abolished CF binding and facilitated native ferritin binding. Further ultrastructural evaluation, using peroxidase labeled LFA lectin, revealed sialic acid moieties in patches on the CAM endothelium. Thus, in early chick embryogenesis, the CAM endothelium displays a continuous pattern of luminal anionic sites comprised in part of sialic acid. As the CAM ages, endothelial anionic sites become reduced. That the expression of endothelial anionic domains remained constant despite changes in CAM microvascular permeability in early development serves to suggest a minimal role for anionic domains in the development of microvascular permselectivity during normal angiogenesis.
Microvascular Research, 1993
Microvascular Research, 1996
During angiogenesis in the chorioallantoic membrane (CAM) of the chick, capillary proliferation o... more During angiogenesis in the chorioallantoic membrane (CAM) of the chick, capillary proliferation occurs primarily by intussusceptive growth. Previously, we reported that such growth in the CAM proceeded without substantial macromolecular extravasation. Neovascularization involving capillary sprout formation, on the other hand, has been associated with a concomitant loss of endothelial selectivity. Thus, the present study tested the hypothesis that endothelial selectivity during angiogenesis is dependent on the mode of microvascular growth. Capillary sprout formation occurs in peripheral regions of the CAM, in addition to the more centrally located areas of intussusceptive growth. In this study, angiogenic endothelial permselectivities were evaluated in these respective areas of CAM microvascular growth by intravital fluorescent microscopy of a graded series of FITC-dextrans. In both cases, the angiogenic endothelia restricted extravasation of macromolecules §20 kDa. Furthermore, capillary sprout endothelia, like the intussusceptive CAM endothelia, remained tightly sealed at the junctional clefts. Thus, angiogenic endothelial permselectivity in the CAM is not dependent on the mode of microvascular growth. Whether distinct cellular mechanisms are operable in capillary endothelial sprouts of the CAM, relative to those of other proliferating sprout endothelia, remains to be tested. ᭧
Microvascular Research, 1995
The Faseb Journal, Apr 1, 2010
This study examined the effects of acute administration of bleomycin (BLM) on lung liquid and pro... more This study examined the effects of acute administration of bleomycin (BLM) on lung liquid and protein exchange in anesthetized sheep prepared with caudal mediastinal lung lymph fistulas. Six sheep received BLM (15 U IV) after a baseline period, while seven others were given BLM after a steady state was obtained following elevation of left atrial pressure (PLA), a procedure intended to minimize changes in pulmonary microvascular surface area and produce high lung lymph flow (QL). Plasma and lung lymph angiotensin converting enzyme (ACE) activities were measured in order to independently assess the effects of BLM on pulmonary endothelial integrity. QL rose significantly in all animals following BLM. The ratio of lymph to plasma protein concentration (CL/CP) did not change in the group given BLM alone, and fell continuously during the period of PLA elevation after BLM in that group. Plasma and lung lymph ACE activities were unchanged following BLM administration in either group. The ultrastructure of the gas-exchanging region of lungs from animals in each group was examined by transmission electron microscopy. The data suggest that acute administration of a low dose of BLM does not increase pulmonary microvascular permeability, but may induce an increase in perfused pulmonary microvascular surface area responsible for increased QL.
Journal of Vascular Research, 1993
Cultured canine jugular vein endothelial cells were seeded on polycarbonate filters to create an ... more Cultured canine jugular vein endothelial cells were seeded on polycarbonate filters to create an in vitro permeability assay. The calculated diffusive permeability coefficient for FITC-BSA across untreated monolayers was 1.1 +/- 0.4 x 10(-6) cm/s. After 15-min incubations with either histamine or bradykinin, the resistance to albumin flux across the monolayers was reduced significantly. Diffusive albumin permeability coefficients were 3.4 +/- 1.8 x 10(-6) and 4.1 +/- 2.0 x 10(-6) cm/s, respectively. Ultrastructural morphometric analyses of the endothelial cell monolayers served to define uniform dimensions of intercellular clefts and similar plasmalemmal vesicle densities in the untreated and the vasomediator-activated monolayers. These results are consistent with the interpretation that the vasomediator-activated pathway across the venous endothelial monolayers is not dependent on sustained intercellular gap formation or sustained expansion of the plasmalemmal vesicle population for the 15-min observation periods. Whether the increased albumin flux is dependent on transient gap formation or on physical changes within the venous endothelial cell glycocalyx remains to be tested.
Objective: To test the hypothesis that stimulation of mast cell deganulation during normal angiog... more Objective: To test the hypothesis that stimulation of mast cell deganulation during normal angiogenesis in the chick chorioallantoic membrane (CAM) serves to acutely activate macromolecular transendothelial pathways.
Microvascular Research, Mar 31, 1997
The American review of respiratory disease
The Anatomical record, 1988
This study presents a quantitative description of ultrastructural changes associated with focal h... more This study presents a quantitative description of ultrastructural changes associated with focal histodifferentiation of the chick chorioallantoic membrane (CAM) microvasculature. Early vessels (4-8 days of a 21-day incubation period) were lined by a thin, but continuous endothelium which was characterized by punctate appositional contacts, an incomplete or absent basal lamina, and few plasmalemmal vesicles. During this stage, there was little ultrastructural distinction between CAM arterioles, capillaries, and venules. During the intermediate stage (9-13 days), the arteriolar endothelium displayed more extensive junctional apposition. In addition, the interstitial (periendothelial) space contained more collagen fibrils and increased cell volume densities, some of which resembled presumptive smooth muscle cells. In contrast to the arterioles, endothelial appositions of the venules and capillaries remained punctate during the intermediate stage. The interstitial space of the venules d...
The Anatomical Record, 1995
The chorioallantoic membrane (CAM) of the chick embryo expands during embryogenesis to meet the i... more The chorioallantoic membrane (CAM) of the chick embryo expands during embryogenesis to meet the increased oxygen demands during growth and differentiation. Temporal and spatial glycosylation patterns of CAM ectodermal and endodermal proteins likely contribute to differentiation of the functional attributes of the CAM. Using lectins for light and electron microscopic observations, we studied the patterns of glycoconjugate expression on the ectoderm and endoderm of the chorioallantoic membrane (CAM) of the chick at days 4.5, 5.0, 5.5, 6.0, and 10 of morphogenesis. For light microscopy, samples of unfixed CAM were incubated with the following FITC lectins: Con A, DBA, GSA-I, GSA-II, PNA, SBA, UEA-I, and WGA. All lectins, except GSA-I and -II, gave positive results. The positive lectins, labeled with HRP, served to ultrastructurally localize PNA, SBA, and WGA, but not DBA binding to the luminal surface of the endoderm. UEA-I and Con A bound similarly except on day 10 when UEA-I no longer bound. On the ectodermal surface, only WGA bound at all times studied. PNA and SBA binding were present from days 5.0 to 6.0 but absent at days 4.5 and 10. DBA binding occurred through day 5.0 but was absent thereafter. UEA-I bound to the ectoderm at days 4.5, 5.0, and 10 but not days 5.5 and 6.0. Con A bound only on days 5.0 and 10. That the ultrastructurally similar ectoderm and endoderm of the CAM display functional differences conforms to the hypothesis that differential expression of glycoconjugate microdomains likely contributes to such functional specialization.
Annals of Vascular Surgery, 1992
Repair of occult arterial injuries is advocated to prevent thrombosis, arteriovenous fistula, and... more Repair of occult arterial injuries is advocated to prevent thrombosis, arteriovenous fistula, and pseudoaneurysm formation. However, recent clinical series describe the healing of arterial intimal injuries and recommend nonoperative therapy. To investigate the arterial wall response to intimal injury, we created intimal flaps in 46 canine femoral arteries. The intimal flaps were imaged by arteriography, angioscopy, and intravascular ultrasound acutely, and at one and three weeks and five months post-injury. Lumen area was measured using caliper techniques (arteriography) and computerized video planimetry (angioscopy, intravascular ultrasound). Intimal and medial thickness were measured by intravascular ultrasound prior to harvest for histologic evaluation by light microscopy. Analysis of 32 patent arteries was performed after exclusion of 14 thrombosed arteries. Residual lumen area (mm 2) correlated closely among the imaging modalities at one week (8.7 +-1.1, 7.3 _ 2.0, 6.9 +-1.8), three weeks (4.2 +_ 0.9, 2.9 +_ 1.0, 2.7 _ 0.8), and five months (5.3 -+ 0.9, 5.0 -0.5, 5.0 -+ 0.9). Maximal intimal and medial thickness occurred three weeks post-injury, coincident with the maximal reduction in lumen area. Although intimal injuries can cause acute and delayed arterial thromboses, observation may be appropriate in selected cases. The evaluation of those patients chosen for nonoperative therapy should extend beyond three weeks, as this is the time of maximal arterial wall response with a continued potential for adverse clinical events. (Ann Vasc Surg 1992;6:50-54).
Respiration Physiology, 1993
We examined the effects of acute expansion of extracellular fluid volume (ECFV) on lung fluid bal... more We examined the effects of acute expansion of extracellular fluid volume (ECFV) on lung fluid balance and the ultrastructure of the pulmonary air-blood barrier in chickens (Gallus domesticus). We compared changes in extravascular lung water content (EVLW) to the sum of mean pulmonary capillary blood pressure and plasma protein osmotic pressure (nc), as a measure of net intravascular filtration pressure (NIFP), produced by graded infusion of avian Ringer's solution. NIFP increased with each volume load largely as a result of decreased nc resulting in progressive increase in EVLW. Progressive interstitial edema occurred with fluid accumulation restricted to the inter air capillary septa, sparing the gas exchanging regions of the air-blood barrier. This was associated with increased thickness of the septa and increased pulmonary capillary endothelial vesiculation. The effect of increased ECFV on pulmonary hemodynamics and lung fluid balance in Gallus is similar to that in mammals.
Microvascular Research, 1996
Using shell-less cultures of chick embryos, the effect of mast cell degranulation on normal rates... more Using shell-less cultures of chick embryos, the effect of mast cell degranulation on normal rates of angiogenesis in the chorioallantoic membrane (CAM) was recorded from Day 6 to Day 9. Based on the centripetal ordering method with capillaries as the initial point of reference, the first two orders of pre-and postcapillary microvessels were evaluated. After infusion of compound 48/80 into the allantoic cavity, the normal proliferation rate of order-1 pre-and postcapillaries was markedly increased. Increases of second-order vessel numerical densities (number/cm 2 ) were less than those of the order-1 vessels. Thus, length densities (mean length 1 numerical density) were increased for the order-1 vessels only. During the accelerated rate of CAM angiogenesis, endothelial restriction of FITC-dextran 150 remained uniformly high. Hence, secreted mast cell products likely worked synergistically with endogenous CAM angiogenic factors between Days 6 and 9, without increasing rates of macromolecular extravasation. However, histamine injection into the allantoic cavity at Day 10, in conjunction with calcium supplementation of the shell-less cultures at Day 9, elevated the rate of FITC-dextran 150 extravasation. Whether differentiation of this transendothelial pathway is required for enhancement of CAM angiogenesis after Day 10 remains uncertain. ᭧
The Anatomical Record, 1996
Background: Intraalveolar bubbles and bubble films have been shown to be part of the normal alveo... more Background: Intraalveolar bubbles and bubble films have been shown to be part of the normal alveolar architecture in vivo from birth through the first 2 days of extrauterine life of rabbit pups (Scarpelli et al., 1996a. Anat. Rec. 244:344-357). The intraluminal boundary between airway free gas and alveolar bubbles at the level of respiratory bronchioles is established within 1 hour after birth. We now examine the lung through the rest of development, namely, 2 weeks, 1,2, and 3 months, and adulthood.
The Anatomical Record, 1997
Background: Having confirmed (Scarpelli et al. 1996. Anat. Rec. 244:344-357 and 246:245-270) the ... more Background: Having confirmed (Scarpelli et al. 1996. Anat. Rec. 244:344-357 and 246:245-270) the discovery of intraalveolar bubbles and films as the normal anatomical infrastructure of aerated alveoli at all ages, we now address three questions. Why have these structures been so elusive? Visible in fresh lungs from the in vivo state, can they be preserved by known laboratory methods? Can they be preserved intact for study in tissue sections?
Tissue and Cell, 1995
The present study served to determine the extent of microvascular endothelial differentiation dur... more 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). CAM's, 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.
Tissue and Cell, 1996
It is generally accepted that luminal surfaces of adult microvascular endothelia present an anion... more It is generally accepted that luminal surfaces of adult microvascular endothelia present an anionic barrier that limits passage of anionic macromolecules. To assess the ontogeny of the barrier, temporal and spatial expression of endothelial anionic sites was evaluated in the chorioallantoic membrane of chicken embryos from days 4.5 to 18 of incubation. After an initial flush, the vessels were perfused with cationic ferritin (CF, 1.0 mg/ml in PBS) for 2 min. Following a second flush to remove unbound CF, the chick chorioal-Iontoic membranes (CAMs) were fixed and processed for electron microscopy. Continuous CF binding was revealed on the luminal endothelium, the junctional clefts and the plasmalemmal vesicles from days 4.5 to 14. However, by day 18, anionic sites had become discontinuous. Prior perfusion with protamine sulfate abolished CF binding and facilitated native ferritin binding. Further ultrastructural evaluation, using peroxidase labeled LFA lectin, revealed sialic acid moieties in patches on the CAM endothelium. Thus, in early chick embryogenesis, the CAM endothelium displays a continuous pattern of luminal anionic sites comprised in part of sialic acid. As the CAM ages, endothelial anionic sites become reduced. That the expression of endothelial anionic domains remained constant despite changes in CAM microvascular permeability in early development serves to suggest a minimal role for anionic domains in the development of microvascular permselectivity during normal angiogenesis.
Microvascular Research, 1993
Microvascular Research, 1996
During angiogenesis in the chorioallantoic membrane (CAM) of the chick, capillary proliferation o... more During angiogenesis in the chorioallantoic membrane (CAM) of the chick, capillary proliferation occurs primarily by intussusceptive growth. Previously, we reported that such growth in the CAM proceeded without substantial macromolecular extravasation. Neovascularization involving capillary sprout formation, on the other hand, has been associated with a concomitant loss of endothelial selectivity. Thus, the present study tested the hypothesis that endothelial selectivity during angiogenesis is dependent on the mode of microvascular growth. Capillary sprout formation occurs in peripheral regions of the CAM, in addition to the more centrally located areas of intussusceptive growth. In this study, angiogenic endothelial permselectivities were evaluated in these respective areas of CAM microvascular growth by intravital fluorescent microscopy of a graded series of FITC-dextrans. In both cases, the angiogenic endothelia restricted extravasation of macromolecules §20 kDa. Furthermore, capillary sprout endothelia, like the intussusceptive CAM endothelia, remained tightly sealed at the junctional clefts. Thus, angiogenic endothelial permselectivity in the CAM is not dependent on the mode of microvascular growth. Whether distinct cellular mechanisms are operable in capillary endothelial sprouts of the CAM, relative to those of other proliferating sprout endothelia, remains to be tested. ᭧
Microvascular Research, 1995