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Am J Physiol Heart Circ Physiol 286: H346-H353, 2004. First published September 25, 2003; doi:10.1152/ajpheart.00772.2003
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Absence of OX-43 antigen expression in invasive capillary sprouts: identification of a capillary sprout-specific endothelial phenotype

Christopher R. Anderson, Ana M. Ponce, and Richard J. Price

Department of Biomedical Engineering, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908

Submitted 12 August 2003 ; accepted in final form 18 September 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Endothelial cells exhibit a number of unique phenotypes, some of which are angiogenesis dependent. To identify a capillary sprout-specific endothelial phenotype, we labeled angiogenic rat mesentery tissue using a microvessel and capillary sprout marker (laminin), selected endothelial cell markers (CD31, tie-2, and BS-I lectin), and the OX-43 monoclonal antibody, which recognizes a 90-kDa membrane glycoprotein of unknown function. In tissues that were stimulated through wound healing and compound 48/80 application, double-immunolabeling experiments with an anti-laminin antibody revealed that the OX-43 antigen was expressed strongly in all microvessels. However, the OX-43 antigen was completely absent from a large percentage (>85%) of the capillary sprouts that were invading the avascular tissue space. In contrast, sprouts that were introverting back into the previously vascularized tissue retained high levels of OX-43 antigen expression. Double-labeling experiments with endothelial markers indicated that the OX-43 antigen was expressed by microvessel endothelium but was absent from virtually all invasive capillary sprout endothelial cells. We conclude that the absence of OX-43 antigen expression marks a novel, capillary sprout-specific, endothelial cell phenotype. Endothelial cells of this phenotype are particularly abundant in capillary sprouts that invade avascular tissue during angiogenesis.

angiogenesis; neovascularization; wound healing; inflammation

ANGIOGENESIS, the formation of new microvessels from existing microvessels, is an essential component of many pathological conditions, including wound healing (20, 21), inflammatory arthritis (33), and tumor growth (3, 9). Depending on the condition at hand, angiogenesis can either be a great benefit or a considerable detriment to a successful clinical outcome. During wound healing, the invasion of new blood vessels into the wound space enhances the supply of oxygen to regenerating tissue (20), thereby facilitating wound closure. In contrast, during tumor growth, aberrant angiogenesis enables tumor enlargement and subsequently provides a gateway to the systemic circulation for metastatic cancer cells (3, 9). Clearly, given the central role of angiogenesis in these and many other conditions, the rational control of blood vessel growth is a logical focus for targeted therapeutic interventions. However, as with most targeted intervention strategies, a crucial challenge is the discovery of tissue-, organ-, and cell-specific molecular targets that permit the delivery of drugs, genes, or other agents to either selected regions of the body or distinct cell populations.

In the context of angiogenesis therapy, endothelial cells are an attractive target because of their primary role in capillary sprouting. Indeed, endothelial cells exhibit several phenotypes that may be exploited for the purpose of site selectivity. At the tissue and organ levels, distinct endothelial phenotypes have been identified in the brain (14), kidney (8), and lung (5). It is likely that many of these organ-specific phenotypes are tightly regulated by cells in the surrounding milieu (1) and exist to perform specialized functions for the organ itself. Of particular interest in the present study, endothelial cells in the brain do not express the antigen for the OX-43 antibody, which is otherwise ubiquitously expressed on endothelial cells (27). However, these same cells do express the transferrin receptor, which is the antigen for the OX-26 antibody in rats (14). Brain-specific endothelial cell expression of the transferrin receptor has been utilized to demonstrate successful transport across the blood-brain barrier using OX-26 antibody constructs (17).

At the level of the microvascular network, vessel-specific phenotypes have also been widely demonstrated (7, 16, 31, 34). Perhaps the most widely studied microvessel-specific endothelial cell phenotype is that of the high endothelial venules, which express adhesion molecules for mediating leukocyte rolling and firm adhesion (31). These molecules, which may be upregulated by either biochemical or hemodynamic factors, include ICAM-1, VCAM-1 (12), and both E- and P-selectin (7). Von Willebrand factor expression also exhibits vessel-specific expression, with marked expression in the venules and diminished expression in the arterioles (1, 32). Additionally, during embryonic development, the Eph-B4 receptor marks the venous endothelium, whereas the ephrin-B2 ligand marks an arteriolar endothelial phenotype (34).

Angiogenesis-specific endothelial cell phenotypes have also been identified. {alpha}v-Integrins are upregulated on endothelial cells in microvessels undergoing angiogenesis (25), and recent advances in both MRI (30)- and ultrasound (19)-based diagnostic evaluation of angiogenic pathologies have utilized contrast agents targeted to {alpha}v{beta}3-integrin. Furthermore, experiments aimed at blocking the function of {alpha}v{beta}3-integrin have shown angiogenesis inhibition (10), indicating that this molecule plays a critical mechanistic role in angiogenesis. More recently, the expression of Thy1.1 by endothelial cells has been shown during adult angiogenesis (18). A functional role for Thy1.1 in angiogenesis has yet to be identified; however, the ability of inflammatory cytokines, but not growth factors, to elicit endothelial Thy1.1 expression highlights the potential importance of inflammation in adult angiogenesis.

In the present study, we used whole mount immunochemistry techniques to uncover a novel endothelial cell phenotype during angiogenesis in response to normal maturation, inflammation, and wound healing. CD31 (PECAM), tie-2, and BS-I lectin were used as endothelial markers, laminin was used as a universal marker of microvessels and capillary sprouts, and differential OX-43 antigen [an ~90-kDa membrane glycoprotein of unknown function (27)] expression was used to identify an endothelial phenotype that was restricted to capillary sprouts.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Angiogenesis models. All animal studies were approved by the Animal Research Committee at the University of Virginia and conformed to American Heart Association Guidelines for the Use of Animals in Research. We studied angiogenesis in whole mounted rat mesentery windows. We defined these windows as the wedge-shaped regions of translucent connective tissue that are bordered by the intestinal wall and ileal blood vessel pairs that run from the mesenteric artery to the intestinal circulation. Because mesenteric windows were whole mounted, the connectivity of the network was preserved, allowing for clear morphological identification of arterioles, venules, capillaries, and capillary sprouts.

Angiogenesis was studied in three separate scenarios. The first was normal development of the animal at both the juvenile and young adult stages. During this natural growth phase, capillary sprouting occurs regularly as microvessel networks gradually infiltrate the previously avascular tissue windows (13, 26). At the young adult and juvenile growth stages, microvessel networks typically occupy the window edges while the central region remains avascular. The second scenario was created using a protocol in which the mast cell-degranulating substance compound 48/80 was dissolved in 0.9% saline (pH 7.2) and administered daily by intraperitoneal injection (24). The compound 48/80 concentration was increased on a daily basis from 100 to 500 µg·ml–1·100 g body wt–1 over a 5-day period. Three days after the final injection, mesenteries were harvested. The final scenario was created by lightly wounding the mesenteric windows. Animals were anesthetized by an intramuscular injection of ketamine (80 mg/kg body wt) and xylazine (8 mg/kg body wt). Under sterile conditions, the mesentery was exposed onto a petri dish. Mesenteric windows were compressed with a cotton-tipped swab, the abdominal wall and skin were sutured closed, and the animal was allowed to recover. Three days after surgery, the mesenteric windows were harvested for immunochemistry and observation.

Tissue harvest and immunochemistry. For tissue harvest, animals were anesthetized as described in Angiogenesis models and subsequently euthanized. Heparinized saline was perfused through a mesenteric artery cannula to remove blood. Several mesenteric windows from each animal were dissected free, washed in cold PBS (pH 7.4), dried on gelatin-coated slides, and fixed in 100% MeOH at –20°C for 30 min.

Immediately after fixation, slides were incubated overnight at 4°C in 0.1% saponin and 5% normal goat serum in PBS (pH 7.4) containing primary antibodies or isolectin. The primary antibodies were monoclonal mouse anti-OX43 antigen (1:100, Serotec), biotinylated monoclonal mouse anti-CD31 (1:200, Serotec and Pharmingen), polyclonal rabbit anti-laminin (1:200, Sigma), and polyclonal rabbit anti-tie-2 (1:1,000, Santa Cruz Biotechnology). The isolectin was biotinylated BS-I lectin (Sigma). When double immunolabeling was performed with primary antibodies from different species (i.e., tie-2/OX-43 or laminin/OX-43) or with BS-I lectin and a primary antibody (i.e., BS-I lectin/OX-43), primary antibodies and/or biotinylated isolectin were simultaneously added in the first incubation step. The next day, the appropriate secondary antibodies (Jackson Immunoresearch) were diluted at a concentration of 1:200 in 0.1% saponin and 5% normal goat serum in PBS (pH 7.4) and applied for 1 h at room temperature. Secondary antibodies were goat anti-IgGs conjugated to either CY2 or CY3. Biotinylated BS-I lectin was revealed by a 1-h incubation in 1:1,000 CY2- or CY3-conjugated streptavidin (Jackson Immunoresearch) at room temperature.

Double immunolabeling with primary antibodies derived from the same species (i.e., OX43/CD31 double labeling) was done with sequential incubations and initiated by the unconjugated primary antibody incubation (OX-43). The next day, CY2- or CY3-conjugated Fab fragments of goat anti-mouse IgG (Jackson Immunoresearch) were used to label the first primary antibody. Fab fragments, which contain only a single binding site, were used instead of whole goat IgG molecules to eliminate a cross-reaction created by binding of the second primary antibody (monoclonal mouse anti-CD31) to the first secondary antibody (goat anti-mouse IgG). This step was followed by a 1-h room temperature incubation in biotinylated anti-CD31. Biotinylated anti-CD31 was revealed by a 1-h incubation in 1:1,000 CY2- or CY3-conjugated streptavidin at room temperature.

Specimen analysis. Specimens were examined with a Nikon TE-300 inverted microscope with both dry (x4 and x10) and oil immersion (x20 and x60) objectives. All confocal images were acquired using a Bio-Rad MicroRadiance scanner attached to the Nikon TE-300 microscope. For each dual-immunolabeling procedure, at least two mesenteric windows were observed per animal. Within these windows, virtually every capillary sprout that could be clearly observed with the microscope was studied. With the use of their anatomic position within the microvascular network as a reference, capillary sprouts were divided into two categories: invading or introverting. By our definition, invading sprouts exhibit directed growth into the avascular tissue region of the mesenteric window. Therefore, these sprouts advance the microvascular network into the avascular tissue. Introverting sprouts were defined based on their growth into tissue regions that were already bordered on all sides by existing microvessels. A growing introverting sprout will eventually intersect an intact microvessel, effectively splitting the vascular loop into two loops and increasing microvascular density in tissue regions that have already been vascularized. A graphic illustration of the anatomic positioning of invasive and introverting sprouts within a mesenteric tissue window is presented in Fig. 1.



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  		Fig. 1. Schematic illustration of the anatomic positioning of invasive and introverting capillary sprouts in a mesenteric tissue window. Avascular and vascularized regions within the wedge-shaped mesenteric tissue window are shown on the left. On the right, the growth of invasive and introverting capillary sprouts into, respectively, the avascular and vascularized tissue spaces are represented.

 


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Angiogenesis in rat mesentery. Figure 2 illustrates representative microvascular networks from the rat mesentery at the time of tissue harvest. Wound healing (Fig. 2c) and compound 48/80-treated (Fig. 2d) mesenteries exhibited a marked increase in vascular density compared with their weight-matched controls (Fig. 2b). Furthermore, compared with both the young adult and compound 48/80-treated mesenteries, arteriolar and venular segments in the wound healing mesenteries were generally characterized by large diameters and relatively short segment lengths. This characteristic morphology was maintained in the capillary plexus, where abundant vessel loops were formed by fairly large-diameter capillaries. In contrast, in compound 48/80-treated mesenteries, arteriolar, venular, and capillary diameters were similar to the young adult controls. As with the wound healing mesenteries, capillary loops were abundant after compound 48/80 treatment and contributed to a considerable portion of the increase in overall vascular density.



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  		Fig. 2. Angiogenesis in the rat mesentery. Confocal microscopy images of CD31-labeled microvascular networks in rat mesenteric connective tissue windows from juvenile (a), young adult (b), wounded (c), and compound 48/80-treated (d) animals are shown. Note the increase in vessel density in the wounded and compound 48/80-treated windows compared with the weight-matched, young adult controls. In a and b, arrowheads denote arterioles and arrows denote venules. Bar = 200 µm.

 

Differential OX-43 expression in microvessels and capillary sprouts. Our first indication that OX-43 antigen expression differed in microvessels and capillary sprouts came from specimens that were immunolabeled with a polyclonal antibody to laminin. Laminin is abundant in the internal elastic lamina of microvessels, and it is also expressed by cells in capillary sprouts (2, 15, 29). Thus, although laminin staining does not distinguish the cells within microvessels and capillary sprouts, it is a marker of both existing and sprouting microvasculature. Confocal images depicting rat mesenteric windows that were double labeled for laminin and OX-43 are shown in Fig. 3. Note that no matter which angiogenic stimulus or maturation phase is observed, both patent microvessels and capillary sprouts are evident with the laminin label. Diffuse laminin staining is also present in the mesothelial layers of the mesentery; however, this does not obscure the underlying microvessels or sprouts. The OX-43 label also clearly denotes microvessels within the networks; however, unlike the laminin label, it begins to diminish at points on the edges of the networks where capillary sprouts arise and begin to invade the avascular tissue space. This is particularly obvious in Fig. 3, c and i, which prominently display invasive capillary sprouts. In most cases, OX-43 antigen expression continues to dissipate along the invasive capillary sprout until it is completely undetectable. Typically, this occurs well before the capillary sprout terminus. As shown in Fig. 3f, this same general pattern of OX-43 antigen expression is often recapitulated in introverting capillary sprouts of the normally developing young adult controls. The OX-43 antigen is also expressed by peritoneal macrophages (27), and this accounts for the punctate interstitial staining evident in Fig. 3.



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  		Fig. 3. Differential OX-43 antigen expression in microvessels and capillary sprouts. Confocal microscopy images of rat mesenteric microvascular networks from compound 48/80-treated (a–c), young adult (d–f), and juvenile (g–i) rats are shown. Microvessels and capillary sprouts were labeled with antibodies to laminin (a, d, and g) and the OX-43 antigen (b, e, and h). In c, f, and i, laminin (red fluorescence) and OX-43 (green fluorescence) images are merged to illustrate that OX-43 antigen expression is absent from many capillary sprouts. Arrows (c and i) denote invasive capillary sprouts. Arrowheads (f) denote introverting capillary sprouts. Bars = 200 µm.

 

This pattern of OX-43 antigen expression in capillary sprouts is quantified in Fig. 4. Figure 4A shows the number of introverting and invasive capillary sprouts that were observed for each angiogenic stimulus. Because some capillary sprouts were not clearly visible due to background fluorescence or overlapping vessel segments, these values do not absolutely represent the total number of sprouts per window in each category. They are, however, generally reflective of the number of sprouts that were present because, on average, only a few sprouts could not be clearly observed per window. Thus, as expected, compound 48/80 and wound healing stimuli generated specimens that allowed for considerably more capillary sprout observations compared with the young adult control. For the juvenile and young adult animals, >75% of the invasive capillary sprout tips were negative for the OX-43 antigen (Fig. 4B). Although not statistically different from the young adult controls, these values were 91% for the wound healing specimens and 87% for the compound 48/80-treated specimens, indicating the prevalence of OX-43 downregulation in invasive capillary sprouts. Figure 4C illustrates these same quantities for the introverting capillary sprouts. Here, a striking difference between the normally developing and the highly angiogenic wound healing and compound 48/80-treated mesenteric windows is evident. In the normally developing animals, the percentages of introverting capillary sprout tips lacking OX-43 antigen expression are almost identical to those seen in the invasive sprouts (~75%). However, compared with the invasive capillary sprouts, the wound healing and compound 48/80-treated mesenteries exhibit a sharp reduction in the percentage of OX-43 negative capillary sprout tips, with these values falling to 15% and 8%, respectively.



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  		Fig. 4. Quantification of the absence of OX-43 antigen expression in capillary sprouts. A: bar graph depicting the mean number of invasive and introverting capillary sprout tip observations made for each group of animals with specimens labeled for both the OX-43 antigen and laminin. B: bar graph illustrating the percentage of invasive capillary sprout tips that are positive for laminin but negative for OX-43 (OX-43–/laminin+). C: bar graph illustrating the percentage of introverting capillary sprout tips that are OX-43–/laminin+. Numbers on x-axis refer to individual and mean animal weights (in g; B and C). Values are means ± SD. *Significantly different (P < 0.05) from adults by unpaired Student's t-test.

 

Microvessel endothelial cells express OX-43 antigen. The results presented in Figs. 3 and 4 illustrate the differential nature of OX-43 antigen expression in microvessels and capillary sprouts. They do not, however, indicate which cells in the sprouts are responsible for generating this expression pattern. Numerous studies indicate that both endothelial cells and pericytes are present in capillary sprouts and that either cell type can precede the other into the tissue during sprouting (2, 22, 23, 28). This implies that endothelial cells, pericytes, or both cell types could generate this expression pattern. Whereas OX-43 antigen expression by pericytes has not been reported, endothelial OX-43 antigen in microvessels is widely recognized (4, 6, 27). We next verified that the OX-43 antigen was expressed by microvessel endothelial cells in our preparations.

To this end, we examined high-magnification confocal images from specimens that were double labeled with the OX-43 antibody and one of three endothelial markers (CD31, tie-2, or BS-I lectin). These results are shown in Fig. 5. Here, the colocalization of the OX-43 antigen with CD31, tie-2 and BS-I lectin is clearly illustrated in microvessel endothelial cells. The vessels shown in Fig. 5 are primarily capillaries; however, the larger-diameter vessel in Fig. 5, d–f, is a collecting venule. It is important to emphasize that the endothelial cells shown in Fig. 5 are not located in capillary sprouts but are instead part of patent microvessel segments that have connections to other microvessels at each end.



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  		Fig. 5. OX-43 antigen is expressed by endothelial cells in microvessel segments. High-magnification confocal microscopy images illustrate that endothelial cells, as defined by CD31 expression (a and d), tie-2 expression (g), and BS-I lectin binding (j), also express the OX-43 antigen (b, e, h, and k) when the endothelial cells are not part of a capillary sprout. Merged color images, indicating the cellular colocalization of the OX-43 antigen with each endothelial marker, are shown in c, f, i, and l. CD31 (a and d) is localized to cell junctions. OX-43 antigen (b, e, h, and k), tie-2 (g), and BS-I lectin (j) are membrane- and/or cytoplasm associated. The large-diameter vessel in d–f is a collecting venule. Arrowheads (c, f, i, and l) denote the absence of staining in endothelial nuclei. Bars = 10 µm.

 

OX-43 antigen is differentially expressed in capillary sprout endothelium. After we verified that microvessel endothelial cells express the OX-43 antigen, we examined OX-43 antigen expression in capillary sprout endothelial cells (Fig. 6). In a pattern similar to that seen with the laminin/OX-43 double-labeled specimens, the OX-43 antigen was present in endothelial cells within the microvascular networks but, with only very few exceptions, was absent from endothelial cells in invasive capillary sprouts. This pattern was sustained in the specimens from the normally developing, compound 48/80-treated, and wound healing groups. Indeed, >90% of the invasive capillary sprout leading endothelial cells in the young adult, wound healing, and compound 48/80-treated groups did not express detectable amounts of OX-43 antigen (Fig. 7B). For the juvenile group, this value was 83%.



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  		Fig. 6. Relative expression of the OX-43 antigen and selected endothelial cell markers in microvessels and capillary sprouts. Confocal microscopy images of rat mesenteric microvascular networks from wound-treated (a–c), compound 48/80-treated (d–f), and young adult (g–l) rats are shown. Microvessels and capillary sprouts are labeled with antibodies to CD31 (a and d) and tie-2 (g) as well as BS-I lectin (j). OX-43 antigen expression is depicted in b, e, h, and k. In c, f, i, and l, images from a–j and b–k are merged to illustrate that OX-43 antigen expression is absent from many capillary sprout endothelial cells. Arrows (c, f, i, and l) denote invasive capillary sprouts. Arrowheads (c) denote introverting capillary sprouts. Bars = 200 µm in c, f, and i and 400 µm in l.

 


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  		Fig. 7. Quantification of differential OX-43 antigen expression in capillary sprout endothelial cells. A: bar graph depicting the mean number of invasive and introverting capillary sprout tip observations made for each group of animals with specimens labeled for both the OX-43 antigen and CD31. B: bar graph illustrating the percentage of invasive capillary sprout tips that are positive for CD31 but negative for OX-43 (OX-43–/CD31+). C: bar graph illustrating the percentage of introverting capillary sprout tips that are OX-43–/CD31+. Numbers on x-axis refer to individual and mean animal weights (in g; B and C). Values are means ± SD. *Significantly different (P < 0.05) from adults by unpaired Student's t-test.

 

Consistent with the laminin/OX-43-labeled specimens, and in contrast to the invasive capillary sprouts, we observed that endothelial cells in the introverting capillary sprouts of the wound healing and compound 48/80-treated mesenteries were often positive for the OX-43 antigen. This is illustrated in Fig. 6c, where a number of short introverting capillaries are seen arising from a venule (arrowheads). Many, but not all, of these introverting sprouts are expressing detectable levels of OX-43 antigen. This result is quantified in Fig. 7C, where in the compound 48/80-treated and wound healing groups, only ~30% of all introverting capillary sprouts were lacking detectable levels of OX-43 antigen. These values were significantly different from the young adult control, in which ~60% of introverting capillary sprouts were negative for the OX-43 antigen.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The primary contribution of this study is the identification of a novel endothelial cell phenotype that is restricted to capillary sprouts. This phenotype, which is marked by the absence of a 90-kDa membrane glycoprotein recognized by the OX-43 monoclonal antibody, was observed during angiogenesis in response to different stimuli, including normal maturation, compound 48/80 application, and wound healing. Of particular importance for targeted therapeutic applications, endothelial cells with this phenotype are particularly abundant in capillary sprouts that are invading avascular tissue.

Organ-specific (5, 8, 14) and microvessel-specific (7, 16, 31, 34) endothelial phenotypes have been widely reported in previous studies. Furthermore, endothelial phenotypes specific to angiogenic microvascular networks have been identified (18, 25). Although these angiogenic phenotypes have been utilized for targeted diagnostic purposes (10, 19), they appear to be expressed ubiquitously throughout the microvasculature of the tissue or organ undergoing angiogenesis. In the present study, we identified an endothelial phenotype that was restricted to capillary sprouts. It is possible that targeting therapeutic agents or diagnostic contrast agents to this smaller subset of angiogenic endothelial cells, particularly those that are invading the tissue, could have significant advantages. For example, it has been recently shown that capillary sprout tip endothelial cells exhibit behaviors distinctly different from endothelial cells in both capillary sprout stalks and microvessels (11). Specifically, capillary sprout tip cells extend filopodia and migrate, but they do not proliferate. Pro- or antimigratory interventions targeted specifically to these invasive endothelial cells could potentially provide a more refined control over angiogenesis.

It is, however, important to note that the phenotype described here is marked by the downregulation, and not the upregulation, of a membrane protein. For this reason, the potential usefulness of this phenotype is currently limited to the isolation of invasive endothelial cells from the organ or tissue undergoing angiogenesis. Once isolated, these cells may be studied in further detail using gene array or proteomic approaches for the identification of specific molecular targets that are upregulated in invasive endothelial cells.

Interestingly, in scenarios in which angiogenesis has been aggressively stimulated through a wounding intervention or compound 48/80 application, this phenotype is seldom recapitulated in capillary sprouts that introvert back into previously vascularized tissue regions. Thus, in addition to being capillary sprout specific, the expression of this endothelial phenotype is also dependent on the spatial position of the capillary sprout within the microvascular network. The reason for this expression pattern is unknown, but a number of factors could be responsible. First, independent of the stimulus, there appears to be a correlation between sprout length and expression of the phenotype. Typically, the invasive sprouts (~90% OX-43–/CD31+ in every group) were longer than the introverting sprouts of the young adult (~60% OX-43–/CD31+), which, in turn, were longer than the introverting sprouts of the compound 48/80-treated and wound healing groups (~30% OX-43–/CD31+). A dependence of the phenotype on sprout length could be explained by the presence of biochemical signals that diffuse from perfused microvessels and mediate OX-43 expression. As the sprout cells move further away from the perfused microvessels, such as would occur with invasive sprouting, the influence of such a factor would diminish. Second, the phenotype could be directly mediated by contact with either blood flow and pressure or other microvessel endothelial cells. For such a mechanism to be operative, OX-43 protein production would cease when the cell pulls away from a patent microvessel to form a sprout or when a new cell is added to the sprout via proliferation. Third, with these complex wound healing and inflammatory stimuli, it is possible that distinct growth factors create the invasive and introverting capillary sprouts, and the expression of the phenotype is dependent on which factor is in effect. Finally, as with many phenotypes that are regulated by cells in the surrounding organ (1), interstitial fibroblasts in the mesenteric connective tissue could modulate the OX-43 antigen phenotype. It is likely that the resident fibroblasts in the vascularized tissue surrounding introverting sprouts are better oxygenated than those in the avascular tissue surrounding invasive sprouts. These local PO2 gradients could create differences in the biochemical signals secreted by these interstitial cells, including any that might mediate OX-43 antigen expression.


   ACKNOWLEDGMENTS
 
GRANTS

This study was supported by National Heart, Lung, and Blood Institute Grant RO1 HL-66307.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. J. Price, Dept. of Biomedical Engineering, Box 800759, Univ. of Virginia Health System, Charlottesville, VA 22908 (E-mail: rprice{at}virginia.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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