Biomedical subjects

D O DeFouw

Publications and source records attributed to D O DeFouw.

At least 19 recordsLinked to original sources

PubMed2000

Restrictive endothelial barrier function during normal angiogenesis in vivo: partial dependence on tyrosine dephosphorylation of beta-catenin.

Differentiation of a restrictive endothelial barrier in the chick chorioallantoic membrane (CAM) occurs between Day 4.5 and Day 5.0 of the normal 21-day gestation. Whether molecular changes in the endothelial cell-cell junctional protein complex contribute to the ontogeny of barrier function represents the principal focus of this study. VE-cadherin has been shown to contribute to the regulation of endothelial cell monolayer permeability in vitro. Accordingly, VE-cadherin is complexed to the cytosolic catenins, and changes in monolayer permeability have been linked to alterations of the cadherin/catenin complex. Currently, a CAM endothelial VE-cadherin/beta-catenin complex was identified, and phosphotyrosine labeling of beta-catenin was decreased concurrently with the abrupt increase in CAM endothelial selectivity between Day 4.5 and Day 5.0. Further, inhibition of protein tyrosine phosphatases impeded regular tyrosine dephosphorylation of beta-catenin at Day 5.0 and this served to partially restore macromolecular extravasation to elevated levels normally present at Day 4.5. Thus, differentiation of selective barrier function in the angiogenic CAM endothelium in vivo is dependent, in part, on tyrosine dephosphorylation of beta-catenin.

Allantois↗

PubMed2000

Vascular endothelial growth factor fails to acutely modulate endothelial permeability during early angiogenesis in the chick chorioallantoic membrane.

The angiogenic endothelium of the chorioallantoic membrane (CAM) offers minimal restriction to macromolecular efflux at Day 4.5 of the normal 21-day chick gestation. Vascular endothelial growth factor (VEGF)-specific Flk-1 and Flt-1 tyrosine phosphorylation was observed at Day 4.5 by receptor immunoprecipitation and requisite immunoblotting. Further, general inhibition of tyrosine phosphorylation by either genistein or tyrphostin (10(-4) M) served to reduce FITC-Dextran 40 extravasation at Day 4.5. Likewise, anti-VEGF, but not anti-FGF-2 mAb, abolished the temporal endothelial hyperpermeability. These results are consistent with the established permeability-enhancing function of VEGF. Normal differentiation of the restrictive CAM endothelial barrier at Day 5. 0 was associated with reduced Flk-1 and Flt-1 expression, but sustained tyrosine phosphorylation of the residual RTKs. Moreover, inhibition of VEGF/RTK activity by anti-VEGF mAb at Day 5.0 did not enhance normal endothelial barrier function. Likewise, neither VEGF (5 x 10(-4) to 10(-15) M) nor PlGF (10(-6) to 10(-8) M), which selectively binds Flt-1, served to increase FITC-Dextran 40 efflux at Day 5.0. Together, these results are consistent with the suggestion that down-regulation of the permeability-related VEGF signal correlates temporally with the ontogeny of restrictive endothelial barrier function during angiogenesis in vivo.

Allantois↗

PubMed2000

Vein interposition cuffs decrease the intimal hyperplastic response of polytetrafluoroethylene bypass grafts.

PURPOSE: The modification of the distal anastomosis of polytetrafluoroethylene (PTFE) bypass grafts with vein interposition cuffs (VCs) has been reported to increase graft patency. However, the mechanisms that are responsible for this improved patency are unclear. Because intimal hyperplasia (IH) is a primary cause of prosthetic graft failure, we hypothesized that VCs affect the distal anastomosis by decreasing the IH response of the outflow artery. METHODS: Twenty-three female domestic Yorkshire pigs (mean weight, 35 kg) underwent 42 femoral PTFE bypass grafting procedures. The PTFE bypass grafts were separated into the following three groups according to distal anastomotic configuration: end-to-side anastomoses (ES), VCs, and cuffs constructed with PTFE (PCs). Four femoral arteries from two pigs served as healthy controls. At sacrifice, the grafts were perfusion fixed, and the distal anastomoses harvested at 1 and 4 weeks. The specimens were hemisected and serially sectioned to identify the heel, toe, and mid-anastomotic regions. The sections were cut into 5-microm segments and analyzed for intima and media thickness and area, intima/media area ratio, and the distribution of IH in the vein cuff. The roles of transforming growth factor-beta1 and platelet-derived growth factor-BB in IH development were assessed with immunohistochemistry. RESULTS: IH development was significantly lower at all areas of the anastomosis, with VCs compared with ES and PCs at 4 weeks (P </=.001). IH decreased in VCs from 1 to 4 weeks in all areas of the anastomosis (P </=.001). PCs showed pronounced IH at the mid-anastomosis as compared with VCs and ES (P </=.001). IH was most pronounced at the toe with ES and PCs (P </=.001). Qualitatively, VCs altered the site of IH development, sparing the recipient artery with preferential thickening of the vein cuff and formation of a pseudointima at the vein-PTFE interface. Immunohistochemistry results showed positive staining for transforming growth factor-beta1, platelet-derived growth factor-BB, and smooth muscle alpha-actin in the hyperplastic intima. CONCLUSION: PTFE bypass grafts with VCs had less IH develop than did grafts with ES and PC anastomoses. IH regression in VCs at 4 weeks suggests compensatory vessel wall remodeling mediated by the presence of the VC. Furthermore, VCs caused a redistribution of hyperplasia to the vein-PTFE interface, delaying IH-induced outflow obstruction in the recipient artery. The marked increase in IH with PCs, despite a similar geometric configuration to VCs, suggests that the biologic properties of autogenous tissue dissipate IH development. Similarly, the flow patterns in PCs and VCs should be identical, which suggests a less important role of hemodynamic forces in VC-mediated protection.

Actins↗

PubMed2000

Differentiation of endothelial barrier function during normal angiogenesis requires homotypic VE-cadherin adhesion.

Endothelial cells express two principal cadherins: VE-cadherin and N-cadherin. We established previously that only VE-cadherin expression was increased during differentiation of barrier function by angiogenic endothelium of the chick chorioallantoic membrane (CAM). Presently anti-VE-cadherin mAb, applied to the CAM at day 4.5 of gestation, served to inhibit the abrupt reduction of macromolecular extravasation that occurs normally at day 5.0. Neither anti-N-cadherin nor nonimmune IgG, on the other hand, prevented this temporal decrease of endothelial permeability. Despite the differential permeability responses, morphometric evaluations defined a reduction of mean paracellular cleft width after the application of either anti-VE-cadherin or anti-N-cadherin. Hence, alteration of molecular sieving characteristics within the junctional clefts, rather than modification of cleft dimensions; likely served as the principal modulator of macromolecular extravasation after inhibition of homotypic VE-cadherin adhesion. These results provide support to the concept that VE-cadherin contributes to the normal differentiation of endothelial barrier function during CAM angiogenesis in vivo.

Animals↗

PubMed1999

Modulation of angiogenic endothelial permselectivity by the cAMP pathway.

The chorioallantoic membrane (CAM) of the chick embryo provides an accessible model of normal angiogenesis in vivo. Previously, we reported a rapid reduction in CAM microvascular permeability to macromolecules between Days 4.5 and 5.0 of the normal 21-day gestation (V. Rizzo et al., 1995, Microvasc. Res. 49, 49-63). Here, we tested the hypothesis that activation of the cAMP signaling pathway at Day 4.5 would acutely increase permselectivity prior to normal differentiation of CAM endothelial barrier properties at Day 5.0. Changes in interstitial optical intensities due to extravasation of a graded series of FITC-dextrans (20, 40, and 70 kDa) were evaluated by computer-assisted image analysis, and endothelial ultrastructure was monitored by transmission electron microscopy. The cAMP analogue 8-bromo-cAMP (10(-4) and 10(-3) M) and forskolin (10(-5) and 10(-4) M), an adenylyl cyclase activator, acutely decreased permeability of the graded FITC-dextran series in a dose-dependent fashion. In addition, the nonspecific phosphodiesterase inhibitor IBMX (10(-4) M) served to increase basal restriction of the 20- and 40-kDa tracers. Further, Rp-cAMPS (10(-4) M), a cAMP antagonist for cAMP-dependent protein kinase, abolished the effects of both 8-bromo-cAMP (10(-3) M) and forskolin (10(-4) M) on FITC-Dextran 40 restriction. In all cases, ultrastructural presentation of both the endothelial cell junctions and the vesicles remained unchanged. The present results are consistent with the concept that exogenous cAMP activation decreased permeability of the angiogenic CAM endothelium at Day 4.5 without concomitant ultrastructural changes in the transendothelial macromolecular exchange pathways. Whether endogenous activity of cAMP contributes to normal differentiation of CAM endothelial barrier properties between Days 4.5 and 5.0 remains to be tested.

1-Methyl-3-isobutylxanthine↗

PubMed1999

Dermal tissue fibrosis in patients with chronic venous insufficiency is associated with increased transforming growth factor-beta1 gene expression and protein production.

PURPOSE: Pathologic dermal degeneration in patients with chronic venous insufficiency (CVI) is characterized by aberrant tissue remodeling that results in stasis dermatitis, tissue fibrosis, and ulcer formation. The cytochemical processes that regulate these events are unclear. Because transforming growth factor-beta(1) (TGF-beta(1)) is a known fibrogenic cytokine, we hypothesized that the increased production of TGF-beta(1) would be associated with CVI disease progression. METHODS: Seventy-eight punch biopsy specimens of the lower calf (LC) and the lower thigh (LT) of 52 patients were snap frozen in liquid nitrogen and stratified into four groups according to the Society for Vascular Surgery/International Society for Cardiovascular Surgery CEAP classification (C, clinical; E, etiologic; A, anatomic distribution; and P, pathophysiology). One set of LC biopsy specimens were analyzed for TGF-beta(1) gene expression with quantitative reverse transcriptase-polymerase chain reaction: healthy skin, n = 6; class 4, n = 6; class 5, n = 5; and class 6, n = 7. A second set of biopsy specimens from the LC and LT were analyzed for the amount of bioactive TGF-beta(1) with a certified cell line 64 mink lung epithelial bioassay: healthy skin, n = 8; class 4, n = 23; class 5, n = 13; and class 6, n = 10. The location of TGF-beta(1) was determined at the light and electron microscopy level with immunocytochemistry and immunogold (IMG) labeling. Multiple comparisons were analyzed with a one-way analysis of variance and the Student-Newman-Keuls post hoc tests. The LC and LT comparisons were analyzed with a two-tailed unpaired t test. RESULTS: The TGF-beta(1) gene transcripts for control subjects and patients in classes 4, 5, and 6 were 7.02 +/- 7.33, 43.33 +/- 9.0, 16.13 +/- 7.67, and 7.22 +/- 0.56 x 10(-14) mol/microg total RNA, respectively. The transcripts were significantly elevated in class 4 patients only (P </=.05). The amount of active TGF-beta(1) in picograms/gram of tissue from LC and LT biopsy specimens as compared with healthy skin biopsy specimens were as follows: healthy skin, <1. 0 pc/g; class 4: LC, 5061 +/- 1827 pc/g; LT, 317.3 +/- 277 pc/g; class 5: LC, 8327 +/- 3690 pc/g; LT, 193 +/- 164 pc/g; and class 6: LC, 5392 +/- 1800 pc/g; LT, 117 +/- 61 pc/g. Differences between healthy skin and the skin of the patients in classes 4 and 6 were significant (P </=.05 and P </=.01, respectively). Differences between the LC and LT biopsy specimens within each CVI group were also significant: class 4, P </=.003; class 5, P </=.008; and class 6, P </=.02. Immunocytochemistry results of healthy skin showed TGF-beta(1) staining of epidermal basal cells only. CVI dermal biopsy results demonstrated positive staining in epidermal basal cells, fibroblasts, and leukocytes. Many leukocytes had positive staining of intracellular granules, which appeared morphologically similar to mast cells. IMG labeling results demonstrated gold particles in the leukocytes and collagen fibrils of the extracellular matrix. CONCLUSION: Our study indicated that activated leukocytes traverse perivascular cuffs and release active TGF-beta(1). Positive TGF-beta(1) staining results of dermal fibroblasts were observed and suggest that fibroblasts are the targets of activated interstitial leukocytes. Increased protein production, despite normal levels of gene transcripts in patients in classes 5 and 6, suggests that alternate mechanisms other than gene transcription regulate protein production. A potential mechanism for quick access and release is storage of TGF-beta(1) in the extracellular matrix. IMG labeling to collagen fibrils support this possibility. Furthermore, TGF-beta(1) was exclusively elevated in areas of clinically active disease, indicating a regionalized response to injury. These data suggest that alterations in tissue remodeling occur in patients with CVI and that dermal tissue fibrosis in CVI is regulated by TGF-beta(1).

Animals↗

PubMed1999

Increased expression of VE-cadherin correlates temporally with differentiation of a restrictive endothelial barrier during normal angiogenesis in vivo.

The purpose of this study was to evaluate temporal expression of VE- and N-cadherins within the angiogenic chick chorioallantoic membrane (CAM). Whether their relative patterns of expression changed in conjunction with abrupt differentiation of the restrictive CAM endothelial barrier between days 4.5 and 5.0 of the 21 days gestation was evaluated. Immunoblots against VE-cadherin depicted an increase of VE-cadherin expression between days 4.5 and 5.0, but no change in expression was detected between days 5.0 and 6.0. N-cadherin expression, on the other hand, remained uniform from day 4.5 to day 6.0. Immunogold-labeled anti-VE-cadherin was found exclusively on the CAM endothelium, and principally along the lateral inter-endothelial junctions. Hence, VE-cadherin expression by the angiogenic endothelium was similar to that of adult endothelium. That VE-cadherin expression by the CAM endothelium was increased between days 4.5 and 5.0 serves to suggest a temporal correlation with the ontogeny of restrictive barrier function in angiogenic endothelium in vivo.

Allantois↗

PubMed1998

Programmed removal of chondrocytes during endochondral fracture healing.

This investigation tested the hypothesis that the removal of chondrocytes during endochondral fracture healing involves an ordered process of programmed cell death. To accomplish this, unilateral closed fractures were created in the femora of 36 Sprague-Dawley rats. The rats were killed in groups of four on days 1, 3, 7, 14, 21, 28, 42, 49, and 56 after fracture. The femora were embedded in paraffin and tested for expression of specific markers of fragmented DNA with use of a terminal deoxyuridyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) technique. To determine the potential for transdifferentiation of chondrocytes to osteoblasts, calluses were also hybridized to detect expression of osteocalcin mRNA. Cell proliferation was assessed by an immunohistochemical detection method for proliferating cell nuclear antigen. A separate group of four rats was killed on day 28 to represent the later stage of the endochondral ossification, and the calluses were examined for cellular morphology with transmission electron microscopy. The results showed a coordination in both time and space of the activities of cellular proliferation and programmed cell death. Cell proliferation was most active in the earlier phases of fracture healing (days 1 through 14), although TUNEL expression was apparent in hypertrophic chondrocytes on day 14 after fracture and persisted until day 28. In the later stages of fracture healing (days 14 through 28), proliferating cell nuclear antigen was no longer synthesized in hard callus (intramembranous bone) and cell removal was the dominant activity in soft callus chondrocytes. Expression of osteocalcin mRNA was detected in osteoblasts but not in hypertrophic chondrocytes or in any other nonosteoblastic cell type. These findings support the hypothesis that the removal of chondrocytes during endochondral fracture healing is part of an ordered transition of tissue types in which the cellular mechanisms are genetically programmed to involve proliferation, maturation, and apoptotic cell death.

Animals↗

PubMed1998

Retinoblastoma protein: a molecular regulator of chronic venous insufficiency.

PURPOSE: Chronic venous insufficiency (CVI) and varicose vein (VV) formation is characterized histologically by the transformation of smooth muscle cells (SMC) from a contractile to a secretory phenotype and by intense collagen deposition. The subcellular regulation point for these processes may be the retinoblastoma protein (pRb), a known inhibitor of cellular proliferation and regulator of differentiation. We hypothesize that pRb phosphorylation is associated with VV formation and functions as a possible subcellular regulator. METHODS: Patients were separated into two groups. Group 1 (n = 6) consisted of vein specimens obtained from patients undergoing coronary artery bypass grafting. Group 2 (n = 6) consisted of patients with symptomatic CVI and duplex confirmed refluxing greater saphenous veins (GSVs) who required GSV stripping. Western blots of GSV protein extracts were performed with anti-human pRb monoclonal antibodies and the degree of nonphosphorylated and phosphorylated pRb was determined. Results were quantified using image analysis of band intensities (computer calibrated intensity units). The ultrastructural appearance of SMCs and the vein wall architecture were qualitatively analyzed with electron microscopy in both groups. RESULTS: Phosphorylated pRb from varicose GSVs exhibited intensities of 523 +/- 188 units, while phosphorylated pRb from normal GSVs demonstrated intensities of 153 +/- 41 units (P < 0.05). SMCs in varicosed GSVs were surrounded by disorganized collagen deposits and displayed a secretory phenotype with spherical vacuolated cells. SMCs from normal GSVs appeared spindle shaped with a purported contractile phenotype and a well-structured extracellular matrix. CONCLUSION: Our data demonstrate that VV formation, in patients with CVI, is associated with phosphorylated pRb and the transformation of SMCs from a contractile to a secretory ultrastructural morphology. The data suggest that SMC dedifferentiation is regulated by pRb and the disinhibition of this protein (phosphorylation) may be an significant factor in the development of lower extremity varicosities.

Blotting, Western↗

PubMed1997

Permselectivity of angiogenic microvessels following alteration of the endothelial fiber matrix by oligosaccharides.

The endothelial glycocalyx, which is composed of integral and peripheral glycoconjugates, forms a fibrous matrix that confers macromolecular sieving properties on the microvascular wall. Changes in pore size within the matrix may regulate macromolecular access to the paracellular and/or vesicular transendothelial pathways. We tested the hypothesis that modifications of the endothelial glycocalyx might play a role in the ontogeny of endothelial permselectivity in proliferating microvessels of the chick chorioallantoic membrane (CAM). Accordingly, we evaluated the effects of Dolichos biflorus agglutinin (DBA) or Arachis hypogaea agglutinin (PNA) lectin binding, and N'N'diacetylchitobiose or hydroxyethyl starch polysaccharide (HES) incorporation on CAM endothelial restriction of FITC-dextrans 40 or 150 at Days 4.5 and 5.0 of development. Extravasation of FITC-dextrans was determined by recording their perivascular interstitial intensities. Following DBA, PNA, and N'N'diacetylchitobiose administration, interstitial accumulation of the tracers near first-order pre- and postcapillaries, and surrounding the capillaries, was similar to that of controls at both Days 4.5 and 5.0. At Day 4.5, pretreatment with HES significantly decreased extravasation of FITC-dextran 40. Thus, retention of HES molecules within the glycocalyx might tighten the matrix, and reduce access of dextran 40 to transendothelial pathways across the angiogenic microvessels.

Animals↗

PubMed1997

Morphometric assessment of the dermal microcirculation in patients with chronic venous insufficiency.

PURPOSE: Ultrastructural assessments of the dermal microcirculation in patients with chronic venous insufficiency have been limited to qualitative morphologic descriptions of venous ulcer edges or venous stasis dermatitis. The purpose of this investigation was to quantify differences in endothelial cell structure and local cell type with emphasis on leukocytes and their relationship to arterioles, capillaries, and postcapillary venules (PCVs). METHODS: Two 4.0 mm punch biopsies were obtained from areas of dermal stasis skin changes in the gaiter region of the leg, as well as from noninvolved areas of skin in the ipsilateral thigh, from 35 patients: CEAP class 4 (11 patients), class 5 (9 patients), class 6 (10 patients), and five normal skin biopsies from patients without chronic venous insufficiency. Electron microscopy was performed on sections at 6700x and 23,800x magnification. At 6700x endothelial cell thickness was determined, and the number of fibroblasts, leukocytes, and mast cells were recorded relative to their proximity to arterioles, capillaries, and PCVs. Similarly, at 23,800x endothelial cell vesicle density, interendothelial junctional widths, and basal lamina thickness (cuff width) were measured. Preliminary evaluation for the presence of transforming growth factor-beta 1 (TGF-beta 1) was performed on three patients using reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Quantitative measurements demonstrated increased mast cell content for class 4 and 5 patients around arterioles and PCVs and increased macrophage numbers for class 6 patients around PCVs (p < 0.05). Fibroblasts were the most common cells observed; however, no differences were demonstrated between groups. No differences were observed in interendothelial junctional widths or vesicle densities in arterioles, capillaries, or PCVs. Basal lamina thickness was increased only at the capillary level (p < 0.05). The results of RT-PCR for TGF-beta 1 messenger RNA were positive in the three patients studied. CONCLUSIONS: Our data suggest that (1) mast cells play a role in the pathogenesis of chronic venous insufficiency; (2) the effects of mast cells, macrophages, or both may be mediated in part by TGF-beta 1; and (3) capillary cuff formation is not associated with widened interendothelial gap junctions, but may be a result of enhanced vesicular transport rate or conformational changes in the interendothelial glycocalyx.

Aged↗

PubMed1996

Intraalveolar bubbles and bubble films: II. Formation in vivo through adulthood.

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 air-way 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. METHODS: In quick succession in anesthetized spontaneously breathing rabbits, the abdominal aorta was transected and trachea was occluded either after an end-tidal exhalation at functional residual capacity (FRC) or after volume expansion in vivo by a single inflation from FRC to 20 or 25 cm H2O pressure (V20, V25). Immediately the thorax was opened and lungs were examined (anterior, anterolateral) through a dissecting stereomicroscope while still in the chest, unperturbed (pleural surface temperature 34 degrees C). Heart and lungs were then removed en bloc and re-examined (anterior, lateral, posterior) to confirm that architecture had not changed (22-27 degrees C). After these immediate examinations, lungs were entered into one of the protocols enumerated in Results. RESULTS: Immediate examination revealed bubbles in all aerated subpleural and deep ("central") alveoli from apex to base at all ages and temperatures. Bubbles were confirmed from two views (top and tangential) and from their individual mobility in response to gentle microprobe pressure. A "common bubble" (> 30 microns to approximately 120 microns inside diameter at FRC) appeared to occupy a single alveolus, sometimes arranged in clusters and collectively accounting for approximately 84% of the total bubble population. Few "large bubbles" appeared to be intraductal. We concluded that "small bubbles" (< or = 30 microns; approximately 16% of the total population) were contracted common bubbles. The free gas-bubble film boundary of the airways was at the level of respiratory bronchioles. Subsequent protocols: (1) Common bubbles moved out of adjoining tissue following subpleural incision. Adjacent bubbles either moved into vacated spaces or into the outside liquid medium. Large bubble(s) followed common bubbles out of the tissue. Small bubbles were less mobile and distal common bubbles did not move. The sequence of bubble movement at V25 was the same. Isolated bubbles had normal surfactant content and surface tension according to "Pattle's stability ratio." Transection revealed analogous conditions in central alveoli. (2) Bubble size increased during inflation from FRC to V25. Airless spaces were aerated with bubbles during inflation. (3) The bubble surface was compressed during deflation to 81% of maximal volume (Vmax) and below, including deflation to minimal volume (Vmin). (4) Bubble/alveolar shape changed from spherical-oval to polygonal when the pleural surface dried at FRC and V25. The original shape was restored when the surface was re-wet. Dry tissue showed but did not emit bubbles when cut; re-wet tissue did. (5) Lung liquid content and volume-pressure were normal at FRC. (6) As expected, conventionally fixed, dehydrated, and embedded sections showed no bubbles. CONCLUSIONS: Bubbles and bubble films are fundamental to normal architecture of aerated alveoli at all lung volumes from birth through adulthood. As infrastructure, they sustain aeration and resist deformation. With ductal films, they may be expected to form an alveolar surface liquid (foam film) network (Scarpelli, 1988. Surfactants and the Lining of the Lung) that modulates liquid balance principally at Plateau borders. They expand and contract respectively during inflation and deflation, maintaining their closed film integrity. Films are compressed to "film collapse" in situ during deflation from volumes well above FRC to Vmin. At these volumes, intact films sustain aeration; some may disperse into t

Age Factors↗

PubMed1996

Capillary sprouts restrict macromolecular extravasation during normal angiogenesis in the chick chorioallantoic membrane.

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 intussuceptive 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 > or = 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.

Allantois↗

PubMed1996

Mast cell activation accelerates the normal rate of angiogenesis in the chick chorioallantoic membrane.

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/cm2) were less than those of the order-1 vessels. Thus, length densities (mean length x 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.

Animals↗

PubMed1996

Distribution of anionic sites on microvascular endothelium of the chick chorioallantoic membrane.

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 chorioallontoic 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 (Rizzo et al., 1995a) serves to suggest a minimal role for anionic domains in the development of microvascular permselectivity during normal angiogenesis.

Animals↗

PubMed1995

Glycoconjugate expression in the chick embryonic chorioallantoic membrane: comparisons of the chorionic ectoderm and allantoic endoderm.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Allantois↗

PubMed1995

Ontogeny of microvascular permeability to macromolecules in the chick chorioallantoic membrane during normal angiogenesis.

Chick embryos were incubated using standard shell-less techniques for microscopic observations at Days 4.5, 5.0, and 5.5 of the normal 21-day gestation. The chorioallantoic membrane (CAM) was prepared for intravital fluorescence confocal microscopy. A graded series of FITC-dextrans (20, 40, 70, and 150 kDa) was injected via vitelline vein. The changes in interstitial optical intensity due to FITC-dextran extravasation were evaluated by computer-assisted image analysis. Apparent permeability coefficients (Ps) were calculated for first order postcapillary vessels from the changes in intensity as a function of time. On Day 4.5, Ps values (means +/- SE x 10(-7) cm/sec) in the CAM microvessels for FITC-Dextran 20, 40, 70, and 150 were 11.8 +/- 1.0, 6.4 +/- 0.4, 3.1 +/- 0.5, and 1.5 +/- 0.5, respectively. The respective Ps values fell dramatically on Day 5.0 to 2.2 +/- 0.5, 0.7 +/- 0.2, 0.6 +/- 0.2, and 0.6 +/- 0.2. On Day 5.5, Ps values for all these FITC-dextrans were equal to 0.7 +/- 0.3. The evaluation of FITC-Dextran 10 on Day 5.5 yielded a Ps value of 1.9 +/- 0.3. Our data demonstrate a rapid reduction in microvascular permeability to macromolecules during normal angiogenesis in the early stages of CAM development. Our data also suggest that these changes in permeability may reflect functional adaptations of the CAM. A comparison of our data to those available in the literature for adult and tumoral tissues demonstrates that microvascular permeability properties in these tissues are different from those of the CAM.

Allantois↗

PubMed1995

Differential lectin binding to microvascular endothelial glycoconjugates during normal angiogenesis in the chick chorioallantoic membrane.

The ontogenesis of endothelial glycoconjugate expression during normal angiogenesis and microvascular development remains unknown. Using intravital fluorescent microscopy, we studied temporal and spatial lectin binding to carbohydrate moieties of luminal microvascular endothelia of the chick embryo chorioallantoic membrane (CAM) during Days 4.5 to 6.0 of the 21-day incubation. We used a battery of eight FITC-lectins (100-200 micrograms/ml). Fluorescent images from precapillary, capillary, and postcapillary segments of the lectin-perfused microvascular units were analyzed by image analysis software to quantitate differences in lectin binding. Results served to indicate a significant decrease in lectin binding to terminal N-acetyl glucosamine, N-acetyl galactosamine, and the N-acetyl galactosamine disaccharide in the glycocalyx of pre- and postcapillary vessels from Day 4.5 to Day 5.0. Lectin binding to N-acetyl glucosamine and N-acetyl galactosamine subsequently increased at Days 5.0 and 6.0. In the capillaries, lectin binding to endothelial galactose, fucose, and sialic acid increased significantly from Day 5.5 to Day 6.0. That these temporal changes in lectin binding to endothelial luminal glycoconjugates coincide with concomitant changes in CAM microvascular permeability (Rizzo et al., in press) serves to suggest a possible association between expression of endothelial glycoconjugates and the ontogeny of microvascular perm-selectivity during normal angiogenesis.

Allantois↗