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Keywords:

  • VEGF;
  • bFGF;
  • tie-2;
  • TGF-β;
  • vasculogenesis;
  • quail embryos;
  • coronary vessels;
  • heart development

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES

Mechanisms regulating coronary vascularization are not well understood. To test hypotheses regarding the influence of key growth factors and their interactions, we studied vascular tube formation (vasculogenesis) in collagen gels onto which quail embryonic ventricles were placed and incubated in the presence of growth factors or inhibitors. Vasculogenesis in this model is dependent on tyrosine kinase receptors, since tube formation was totally blocked by genestein. Tube formation was attenuated when anti-bFGF or anti-VEGF neutralizing antibodies were added to the medium and nearly completely inhibited when the both were added. The attenuation associated with anti-VEGF was due primarily to a decrease in assembly of endothelial cells, while that associated with bFGF was primarily due to a reduction in endothelial cells. Soluble tie-2, the receptor for angiopoietins, also had an inhibitory effect and, when added with either anti-bFGF or anti-VEGF, markedly attenuated tube formation. At optimal doses, tube formation was enhanced 6.5-fold by bFGF and 2.5-fold by VEGF over the controls. Each of these growth factors was dependent upon the other for optimal induction of tube formation, since neutralizing antibodies to one markedly reduced the potency of the other. VEGF potency was also markedly reduced when soluble tie-2 was added to the medium. Tube formation was virtually totally blocked by exogenous TGF-β at doses > 1 ng/ml, while neutralizing TGF-β antibodies enhanced tube formation 2-fold in the 30 ng–30 μg range. These data provide the first documentation of multiple growth factor regulation of coronary tube formation. © 2001 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES

The first step in establishing a coronary vasculature involves the migration of angioblasts from the subepicardium into the myocardium, their differentiation into endothelial cells, and their assembly to form vascular tubes (Mikawa and Fischman, 1992; Poelmann et al., 1993; Rongish et al., 1994; Viragh et al., 1993). This process is termed vasculogenesis and also consists of fusion of the vascular tubes (a process reviewed by Drake and Little, 1999). Previous work in our laboratory has shown that vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF or FGF-2) are temporally and spatially related to vascularization of the rat myocardium during prenatal and early postnatal development (Tomanek et al., 1996, 1999), and that both enhance tube formation when administered in ovo in the chicken (Tomanek, et al., 1998). While these findings clearly implicated these growth factors in myocardial vascularization, they did not define their precise role in the events of vascular tube formation from differentiating endothelial cells.

Because in vivo studies on coronary vasculogenesis face technical limitations, we have utilized explanted embryonic hearts that can be more effectively perturbed with regard to growth factors and/or the environment in which coronary microvessels form (Ratajska et al., 1995; Yue and Tomanek, 1999). In this model, endothelial cells and their precursors migrate from the explants onto a collagen gel and form endothelial-lined tubes, which in quail are visualized by immunostainig with the specific endothelial cell marker QH1 and confocal microscopy (Yue and Tomanek, 1999). Previous work, using this model, has demonstrated the marked stimulatory effect of VEGF165 and hypoxia on vasculogenesis (Yue and Tomanek, 1999). The overall goal of the work described here was to determine the roles of key vasculogenic/angiogenic growth factors on tube formation, the earliest stage of formation of the coronary vasculature. This goal originated from the premise that multiple growth factor regulation in the heart includes interdependency, a concept that the optimal effect of a growth factor on vasculogenesis and/or angiogenesis is dependent on another growth factor or factors acting on some aspect of vessel assembly. Based on our previous work, the first aim of the current study was to test the hypothesis that VEGF and bFGF are interdependent regulators of embryonic coronary vasculogenesis. The second aim of this study was to test the hypothesis that the angiopoietin receptor, tie-2, facilitates coronary vasculogenesis. This hypothesis was based on the rationale that angiopoietins, along with VEGF are major regulators in developmental systems (Hayes et al., 1999; Koblizek et al., 1998; Suri et al., 1996). Finally, we tested the hypothesis that TGF-β1 inhibits coronary vasculogenesis, since in vitro experiments using cell lines have shown it to inhibit endothelial cell growth, migration and capillary formation (Li et al., 2000; Pepper, 1997). However, the role of TGF-β1 in vasculogenesis/angiogenesis is controversial, since this growth factor has been found to induce VEGF expression (Li et al., 1997; Seko et al., 1999; Zheng et al., 1999). Moreover, endoglin, a receptor for TGF-β isoforms 1 and 3, is essential for embryonic development of blood vessels (Arthur et al., 2000).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES

Angioblasts/endothelial cells from cultured embryonic explanted hearts migrate onto collagen gels and form vascular tubes, as previously detailed (Yue and Tomanek, 1999). These cells stain with QH1 antibody and are recognized with confocal microscopy. We refer to the vascular tube formation as vasculogenesis, because the assembly of endothelial cells into tubes is the main event occurring during the time period studied. We assume that the sprouting of vascular tubes or their partitioning (angiogenesis) plays a more minor role. The quantitative assays in our experiments consisted of measurements of (1) aggregate (total) tube length, adjusted for the perimeter of the heart on the gel, and (2) the number of free endothelial cells (those that are not associated with tubes). All explants were cultured for 2 days in M199 plus 10% FBS, then for one day in low serum M199 and then growth factors or inhibitors were added. We assayed tube formation either 48 or 72 hr after adding the reagents. Some minimal tube formation could be seen at 24 hr at the cut surface of the explants. Tubes were always in contact with the epicardial surface of the ventricles, as verified by scanning electron microscopy. By 48 hr, tube density was markedly increased. Using this system we determined the aggregate tube length formed, i.e., the lengths of all tubes and their branches were measured and totaled. Since all ventricular surfaces (circumferences) were not identical, we expressed the data as aggregate tube length per ventricular perimeter.

Coronary Vasculogenesis Is Dependent on Multiple Factors

We first documented that tube formation is dependent upon tyrosine kinase receptors. Addition of genestein to the incubation medium completely and consistently blocked the formation of vascular tubes on the collagen gels (Fig. 1). We then explored the roles of bFGF and VEGF in tube formation by adding monoclonal neutralizing antibodies to the incubation media. Preliminary experiments indicated that addition of 6 μg/ml provided maximal blocking of the endogenous growth factors in our system. Our data revealed that, compared to controls, tube formation was only 22 and 44%, respectively, in the bFGF and VEGF neutralizing antibody treated groups (Figs. 1 and 2). However, the major mechanism by which vasculogenesis was inhibited, as indicated by total tube length formed, differed between the two neutralizing antibodies. Explants treated with bFGF neutralizing antibodies (bFGF-AB) had a 60% reduction in free EC (cells not components of tubes), while the explants treated with VEGF neutralizing antibodies (VEGF-AB) showed no significant reduction in free cells. These data suggest that limitations in tubulogenesis associated with VEGF-AB treatment were due primarily to a limited assembly of EC into tubes, and not to the availability of the cells. In contrast, inhibition of tube formation with bFGF-AB treatment is due, at least in part, to fewer available cells. Addition of soluble tie-2 had a similar effect as that of VEGF-AB. When VEGF-AB and bFGF-AB were added in combination, or either was combined with the soluble tie-2 receptor, tube formation was inhibited by 80 to 90% (Fig. 3).

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Figure 1. Representative confocal images of explants. Top: Explants at 72 hr after treatment with various inhibitors of vasculogenesis. Bottom: Explants at 48 hr after treatment with bFGF and VEGF alone or in combination with neutralizing antibodies or soluble Tie-2. Tube formation and cell migration are seen. Scale bar = 1 mm.

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Figure 2. Inhibition of vasculogenesis by anti-bFGF (6 μg/ml), anti-VEGF (6 μg/ml), or soluble tie-2 receptor 6 μg/ml. Means and standard errors for aggregate (total) tube length and number of free endothelial cells are shown. *Significant differences from control (P ≤ 0.05). The crosshatched areas represent median values. Number of explants: bFGF AB = 29; VEGF AB = 12; Sol. Tie-2 = 33

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Figure 3. Inhibition of vasculogenesis by exposure of heart explants to combination of neutralizing antibodies to bFGF and VEGF, and to soluble tie-2 receptor. Means and standard errors for aggregate (total) tube length and number of free endothelial cells are shown. *Significant difference from controls (P ≤ 0.05). The cross-hatched areas represents median values. Number of explants: bFGF + sol. Tie-2 = 20; VEGF AB + Sol. Tie-2 = 21; bFGF AB + VEGF AB = 7.

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Exogenous VEGF and bFGF Stimulate Rapid and Marked Coronary Vasculogenesis

Having documented the influence of endogenous growth factors on tube formation, we conducted experiments on the efficacy of exogenous bFGF and VEGF alone or in combination (Fig. 4). Using a dose range of 30–600 ng/ml, we found that 30–150 ng/ml of VEGF enhanced maximal tube length formed about 2–3-fold. The 150-ng dose was used in subsequent experiments because the data were based on a large sample size and because the highest individual values were obtained using this dose. High doses (300 and 600 ng) of VEGF failed to enhance tube formation above control values. Exogenous bFGF stimulated tube formation in a dose-dependent manner, with the maximal effect occurring at 150 ng/ml. The effect was more marked, a 6.5-fold increase, than that seen with VEGF. When the two growth factors were administered in combination of doses of 30–300 ng each, tube formation was not significantly enhanced compared to administration of each growth factor alone. Therefore, we did not find the effects of bFGF and VEGF to be additive in our model.

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Figure 4. Tube formation stimulation in heart explants by VEGF and bFGF. Both growth factors stimulate tube formation. A dose-dependent response is evident for bFGF. High doses of VEGF (300 and 600 ng/ml) do not stimulate tube formation. A reduction in effectiveness for bFGF is seen at a dose of 600 mg/ml. When VEGF and bFGF are both added to the culture media, an additive effect is not evident at the most effective individual doses (150/150 ng). Means and SEM are shown; the crosshatched areas represent median values. Each group mean is based on 7–8 explants, except VEGF 150 and bFGF 150, which consist of 47 and 19 explants, respectively.

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Although synergism for bFGF and VEGF was not documented, we tested the hypothesis that the two growth factors are interdependent (Fig. 5). Accordingly, we added bFGF-neutralizing antibodies to cultures stimulated with VEGF protein, and VEGF-neutralizing antibodies to cultures stimulated with bFGF protein. The data shown in Figure 5 indicate that 12 μg/ml of anti-bFGF totally abolishes the effect of VEGF. In contrast, anti-VEGF at 6 or 12 μg/ml markedly reduces, but does not abolish, the effect of exogenous bFGF. These data indicate that while each of these growth factors is dependent on the other for maximal efficacy, VEGF requires the presence of bFGF for its function.

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Figure 5. Tube formation in heart explants by VEGF and bFGF when one of the growth factors is inhibited by neutralizing antibodies. Means and standard errors are shown; the crosshatched bars represent median values. Anti-bFGF completely negates tube formation by VEGF, while anti-VEGF inhibits tube formation due to bFGF by about 55–60%. Each group consists of 7–10 explants.

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VEGF's Effectiveness Is Dependent on Angiopoietins

Having shown that the presence of soluble tie-2 receptor in the explant media has some attenuating effect on tube formation, we tested the hypothesis that the effectiveness of VEGF in stimulating vasculogenesis is most effective in the presence of angiopoietin. The addition of soluble tie-2 receptor to explants that were stimulated with VEGF (Fig. 6) reduced tube formation to levels below the controls and accordingly totally prevented VEGF-associated growth.

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Figure 6. Effects of soluble tie-2 receptor on VEGF-stimulated tube formation in explanted hearts. Means and standard errors are shown; the crosshatched bars represent median values. Each group consists of 8 explants.

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TGF-β1 Inhibits Tube Formation in the Explant Model

The role of TGF-β1 in vasculogenesis in the coronary explant model was evaluated by adding various doses of TGF-β1 protein or TGF-β1 neutralizing antibodies to the culture media. As illustrated in Figure 7, tube formation was nearly completely inhibited by TGF-β1 doses in the range of 150 pg/ml to 150 ng/ml. Anti-VEGF neutralizing antibodies in the range of 30 ng/ml to 30 μg/ml facilitated about a 2-fold increase in tube formation compared to the nontreated controls. These data indicate that TGF-β1 inhibits the formation of coronary vascular tubes in this in vitro system.

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Figure 7. Effects of TGF-β1 on tube formation in heart explants. The effects of various doses of the protein and its neutralizing antibody are shown. Mean and standard errors are provided. The crosshatched areas are median value. Each group consists of 7–8 explants.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES

This study is the first documentation that coronary vasculogenesis is regulated by multiple growth factors and their interactions. Several major findings are indicated from our experimental findings. First, both VEGF and bFGF stimulate coronary vasculogenesis, and each facilitates the potency of the other. Moreover, the potency of bFGF is greater than that of VEGF. Second, angiopoietin, via the tie-2 receptor, facilitates vasculogenesis and enhances the effectiveness of VEGF. Third, TGF-β inhibits vascular tube formation.

The critical role of VEGF in vascular development in the embryo has been well documented (Carmeliet et al., 1996). Moreover, a role for bFGF has also been indicated for angioblast differentiation (Cox and Poole, 2000). Previous work (Mandriota and Pepper, 1997) illustrated that tube formation and plasminogen activator expression of microvascular and aortic bovine endothelial cells is dependent on endogenous bFGF. Some studies using microvascular cell cultures have also shown that the effects of VEGF and bFGF are additive (Goto et al., 1993; Pepper et al., 1992). In this quail heart explant study, we did not see a significant additive effect of these growth factors on tube formation. Similarly, an earlier study from our laboratory using rat embryonic (E12) heart explants found no additive effect on cell number when VEGF and bFGF were added together (Ratajska et al., 1995). This is not so surprising in light of the differences between a culture system containing only endothelial cells and the heart explant model that contains multiple cell types and, therefore, both paracrine and autocrine signaling. Thus, endogenous bFGF and VEGF are already present in a system to which growth factors or inhibitors are added exogenously. Most importantly, our study documents an interdependence of VEGF and bFGF in the embryonic explanted heart, as evidenced by (1) the inability of VEGF to stimulate tube formation when neutralizing antibodies to bFGF are added to the system, and (2) marked limitations in bFGF-stimulated tube formation in the presence of neutralizing VEGF antibodies.

Our finding that VEGF and bFGF are interdependent in their effects on embryonic heart endothelial cells is consistent with data from adult, non-coronary, endothelial cells (Pepper et al., 1998). VEGF-induced tube formation by cultured bovine endothelial cells has been shown to be dependent upon bFGF (Jonca et al., 1997; Mandriota and Pepper, 1997). That study also revealed that the VEGF-dependence on bFGF is not via a decrease in VEGFR-2 (flk-1). The importance of FGF in VEGF signaling has also been described in a glioma cell line (Ryuto et al., 1996). Similarly, bFGF-induced endothelial cell proliferation is inhibited when VEGF neutralizing antibodies are added to endothelial cell cultures (Seghezzi et al., 1998). Furthermore, these neutralizing antibodies markedly reduce bFGF-induced angiogenesis in the cornea. VEGF pontentiates the mitogenic effect of bFGF in vascular smooth muscle (Couper et al., 1997). Thus, both VEGF and bFGF are key angiogenic growth factors during tube formation in the embryonic heart.

Angiopoietins are recognized as important molecules that influence endothelial cell behavior during vessel formation. Angiopoietin-1–deficient mice have a vascular network that is less complex than that of their wild type counterparts (Suri et al., 1996) and angiopoietin-1 has been shown to promote endothelial cell migration, survival, and tube formation (Hayes et al., 1999), and capillary sprouting (Koblizek et al., 1998). Angiopoietin-1 appears to potentiate the effects of VEGF in mouse corneal assays (Asahara et al., 1998). Tie-2, the receptor for this ligand, is essential for vascular development during late organogenesis (Puri et al., 1999), and has been found to mediate angiopoietin-1 induced endothelial cell migration (Kim et al., 2000). Addition of soluble tie-2 to explants attenuated tube formation, and when added with either anti-bFGF or anti-VEGF virtually negated tube formation. By adding the soluble tie-2 receptor to the explant cultures in which tube formation was stimulated by VEGF, we were able to document VEGF's vasculogenic dependence on angiopoietins, since tube formation under this condition could not be enhanced above control values.

The precise role of TGF-β in vasculogenesis is not yet documented, and, as noted in a review by Pepper (1997), may be dictated by a number of variables. Its pro-angiogenic effects are usually indirect and may be mediated by inflammatory cells. In some circumstances, e.g., gastric sarcoma (Saito et al., 1999), it may stimulate angiogenesis by up-regulation of VEGF. TGF-β does not effect endothelial cell proliferation during intimal lesion formation (Smith et al., 1999). Endothelial cell specificity or the local environment may also serve as a determinant of the effects of TGF-β. For example, neutralizing TGF-β antibody infusion in neonatal rats delays in vivo glomerular capillary formation (Liu et al., 1999), while embryonic tissues in TGF-β knockout mice exhibit normal vascular structures in embryonic, but not extraembryonic tissues (Dickson et al., 1995). Data from in vitro studies, using various endothelial cell lines show an inhibitory effect of TGF-β on proliferation (reviewed by Pepper, 1997). However, our explant model, as noted earlier, is not limited to endothelial cells. Therefore, we tested the role of TGF-β in a system that contains the features of both in vivo and in vitro system. Our data support two conclusions. First, exogenous TGF-β1 at a wide range of doses inhibits coronary tube formation. This finding is in concert with previous work that found TGF-β1 to inhibit epidermal growth factor stimulated rat heart endothelial cell growth in monolayer culture (Mooradian and Diglio, 1990). Second, and more important, we have demonstrated that inhibition of endogenous TGF-β accelerates vascular tube formation. Thus, endogenous levels of TGF-β inhibit proliferation and/or migration of endothelial cells in the embryonic heart. One possible explanation of the antagonistic effect of TGF-β1 is that it has been shown to down-regulate angiopoietin-1 (Enholm et al., 1997). Whether TGF-β1 consistently plays an inhibitory role at other time points in coronary vascular developments has not been determined. Evidence that it upregulates VEGF in adult cardiac myocytes subjected to cyclic stretch (Seko et al., 1999) suggests that it may play a proangiogenic role. Thus, generalizations concerning this growth factor's role in angiogenesis are not warranted.

Data from this study provide new evidence that multiple growth factors play a role in the formation of vascular tubes by endothelial cells from the embryonic heart. Although the explant model is an in vitro system, it mimics the in vivo heart because paracrine signaling and cell to cell interactions are not eliminated. Based on our findings, we conclude that at least three growth factors (VEGF, bFGF, and angiopoietin-1) facilitate the events of coronary vascular tube formation and that TGF-β counteracts some event(s) in the vasculogenic cascade. In that these experiments concerned only tube formation (vasculogenesis) in the embryonic heart, the conclusions should not be extrapolated to subsequent events in vascularization of the heart. We recognize that subsequent growth of vascular tubes and the formation of larger vessels may involve somewhat different roles for the growth factors studied in our experiments. Moreover, the contribution of other growth factors and their interactions undoubtedly play important roles in these subsequent events.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES

Embryonic Heart Explant Culture System

At the end of a 6-day incubation period, fertilized quail eggs (Coturnix japonica) were removed, the hearts dissected, and the apical one-third of the ventricles placed on collagen gels in 4-well tissue culture plates with the cut edge in contact with the gel, as previously described (Yue and Tomanek, 1999). Collagen I was prepared from rat tails (Collaborative Research, Bedford, MA). The hearts were incubated for 4–6 hr at 37°C to allow attachment of the explant to the collagen gel and then M199 supplemented with 10% heat-inactivated FBS was added. Two days later the medium was replaced with low serum M199 (heat-inactivated FBS) supplemented with insulin-transferrin-selenium-A (ITS Life Technologies) for one day, and then growth factors or inhibitors were added to the media. The specimens were then studied after either 48 or 72 hr of exposure to the growth factors or inhibitors. Each experiment included controls (nontreated explants); the magnitude of tube formation, or inhibition of tube formation was based on the control values for a given experiment.

We purchased growth factors from commercial sources: VEGF165 (R&D), bFGF (Collaborative Biomedical Products), and TGF-β1 (Sigma). Polyclonal neutralizing antibodies to TGF-β1 were purchased from R&D. Monoclonal neutralizing antibodies to VEGF and bFGF were a kind gift of Dr. Tommy Brock (Texas Biotechnology Corporation, Houston, TX). The soluble form of the extracellular domain of murine tie-2 (ExTek.6His) was developed as previously described (Lin et al., 1997).

Confocal Microscopy and Assessment of Vascular Tube Formation

As previously detailed (Yue and Tomanek, 1999), the gels containing the vascular tubes and explanted hearts were fixed overnight in 4% paraformaldehyde in PBS at 4°C. After washing in PBS, nonspecific binding was blocked by incubation in 1% bovine serum and 0.5% Triton-X100 in PBS. We then incubated the samples in QH1 antibody (Hybridoma Bank, University of Iowa) for 3 hr at room temperature. QH1 binding was detected by fluorescein isothiocyanate conjugate-labeled goat-anti-mouse secondary antibody (Sigma). Confocal microscopy and BIORAD laser sharp software were employed to scan the samples. After capturing images of the explants, vascular growth was quantified by measuring the total length of tubes formed in the gel per perimeter of the ventricular explant using Image Pro Plus software (Media Cybernetics). The number of “free” endothelial cells/ventricular perimeter (i.e., those not components of tubes) was also determined. All measurements were done in a blinded manner.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES
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