INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE FORMATION OF FUNCTIONAL blood vessels during embryogenesis requires the assembly of endothelial and vascular smooth muscle cells (VSMC) (1, 2, 4, 13, 16). The initial steps of this process involve the formation of a new endothelial network through vasculogenesis, remodeling of this primitive network and subsequent branching angiogenesis (16). As part of the remodeling process, VSMC migrate toward these sites and align around the endothelial cells to form a multilayered vessel wall. The recruitment of VSMC and further interactions between the two cellular layers of the nascent vasculature are mediated through secreted signaling molecules and extracellular matrix (ECM) constituents. Components of the ECM between the endothelial and VSMC layers are products of both cell types and exert their regulatory functions through both paracrine and autocrine pathways. Osteopontin (Osp) and vitronectin (VN) are examples of ECM adhesion molecules with known regulatory functions during blood vessel development and remodeling (11, 18). These glycoproteins are expressed by endothelial and VSMC, contain arginine-glycine-aspartic acid (RGD) adhesion motifs, and transmit their functions through VN receptors (19).

Del1 represents a regulatory ECM protein that is expressed by endothelial cells during early embryogenesis, with its expression being downregulated later in development (6). Similar to Osp and VN, Del1 contains an active RGD sequence that mediates the binding and migratory response of endothelial cells through interaction with the _v3-integrin receptor (6, 15). Unlike Osp and VN, Del1 has been shown to be angiogenic in the chorioallantoic membrane assay, suggesting the activation of additional, growth-factor-like signaling pathways (15). Furthermore, Del1 appears to be one of a number of epidermal growth factor (EGF) repeat containing genes expressed in the vascular wall. The recently characterized genes developmental arteries and neural crest epidermal growth factor-like (DANCE) and embryonic vasculature and epidermal growth factor-like repeats (EVEC) have EGF repeats as well as RGD motifs (8, 12). Expression of these two genes has been shown to be upregulated in VSMC after injury, raising the hypothesis that they are involved in the response of this cell type to vascular wall disease. To determine whether Del1 might also be activated in the setting of vascular injury and interact with VSMC through their _v3-receptors, the current studies were performed. They suggest that Del1 represents a novel substrate for VN receptors on this cell type and that it has autocrine and paracrine functions that can direct vascular wall development and subsequent vascular remodeling.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Expression of recombinant Del1 proteins in Baculovirus. A murine Del1 major cDNA clone containing a His tag at the carboxy terminus was cloned into the shuttle vector pACGP67B and transfected into Sf9 cells with Baculogold reagents (Pharmingen; San Diego, CA), and recombinant protein was purified and evaluated as previously described (6, 15). Del1 protein was kept frozen at 450 µg/ml.

Immunochemicals and reagents. Synthetic peptides GRGDSP (GRD), GRGESP (RGE), and GPenGRGdsPCA (Pen RGD) were purchased from GIBCO-BRL. Peptides were dissolved in Dulbecco's phosphate-buffered saline (DPBS) at 20 mg/ml and kept frozen. Human VN (GIBCO-BRL) was resuspended and kept frozen in DPBS at 100 µg/ml. Del1 protein was kept frozen at 450 µg/ml. Monoclonal antibodies to _v3- (LM-609) and _v-integrins were purchased from Chemicon (Temecula, CA). A monoclonal antibody to anti-human ₃-integrin (clone AP-3) was a generous gift from Dr. Peter J. Newman (The Blood Center of Southeastern Wisconsin). Monoclonal antibodies to vinculin, smooth muscle -actin, and fluorescein isothiocyanate-phallicidin were purchased from Sigma (St. Louis, MO). Polyclonal anti-p125FAK was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Cy5-conjugated goat anti-rabbit and anti-mouse antibodies used for immunofluorescence studies were purchased from Jackson Immunoresearch (West Grove, PA).

Cell culture. Human aortic VSMC were a generous gift of Dr. Phillip Tsao (Stanford University; Stanford, CA). VSMC were maintained and passaged into Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) (Collaborative Research; Bedford, MA), 100 U/ml of penicillin, and 100 µg/ml of streptomycin. For all experiments, only cell passages 3-5 were used. For serum-free (SF) experiments, low-passage VSMC were weaned over 48 h into media containing 1% FBS before the experiment. The cells were then washed two times into DPBS, immobilized as described above, and plated over protein coated wells in SF DMEM.

Cell adhesion assay. Cell adhesion assays were performed as described by Leavesley et al. (9). Briefly, 96-well plates (Nunclon, Nunc; Naperville, IL) were coated at 4°C overnight with each ligand (200 µl at 20 nM in DPBS). Before the cells were added, wells were washed three times with SF DMEM. VSMC were harvested with limited trypsin-EDTA treatment, washed twice, and cultured over each substrate at 5 × 10⁴ cells/well (in triplicates) in SF DMEM containing 1% BSA. At each time point, the wells were washed twice with warm DPBS to remove nonadherent cells. The remaining cells were then fixed with 4% paraformaldehyde (PFA) in DPBS (pH 7.4) for 10 min at room temperature and stained with 0.5% toluidine blue for 5 min. The wells were rinsed with water, cells were solubilized with 0.1% sodium dodecyl sulfate (SDS), and the optical density was determined at 595 nm on a Bio-Rad microtiter reader. Synthetic blocking peptides and antibodies (50 µg/ml) were preincubated with cells for 30 min at 37°C before their addition to the wells. Cycloheximide studies were performed by pretreatment of VSMC for 2 h before and during the experiments with cycloheximide (25 µg/ml).

Cell migration assays. Migration assays were performed with a modification to the protocol described by Leavesley et al. (10). Substrates were diluted to 50-100 nM in SF-DMEM containing 200 µg/ml BSA and placed either in the bottom or top well of a modified Boyden chamber (8.0-µm membrane, Transwell, Costar). VSMC were harvested with limited trypsin-EDTA treatment, washed with SF DMEM, and resuspended in the SF DMEM and added in 200 µl (3-4 × 10⁴ cells) to the upper chamber in the presence or absence of synthetic peptides (100 µM) or monoclonal antibodies (50 µg/ml). Migration was measured after 6- to 8-h incubation at 37°C. The cells were fixed and stained with 3% PFA-toluidine blue. The cells were washed with water, and the cells on top were removed with the use of a cotton swab. The cells that had migrated through the membrane were then counted from multiple fields. Experiments were performed in quadruplicate.

Cell proliferation assay and proliferating cell nuclear antigen staining. Proliferating cell nuclear antigen (PCNA) assays were performed according to the manufacturer's protocol with the CellTiter 96Q kit (Promega; Madison, WI). Briefly, 5 × 10⁴ cells were plated onto 96-well microtiter wells in triplicate. At each time point, an equal volume of the CellTiter 96AQ solution was added to each well. Optical density was determined after 1 h and again at 4 h of incubation at 37°C with the use of a 490-nm filter on an automated microtiter reader. PCNA staining was performed as per the manufacturer's protocol (Boehringer-Mannheim; Indianapolis, IN).

Apoptosis assay. TdT-mediated dUTP nick-end labeling (TUNEL) assays were performed according to the manufacturer's recommended protocol for the In Situ Cell Death Detection Kit (Boehringer-Mannheim). Briefly, 2 × 10⁴ cells were cultured in multiwell chamber slides (Nunc), which were precoated with soluble substrate overnight at 4°C. Cells were either cultured in SF DMEM or in the presence of 10% serum (positive control). At each time point, cells were washed with DPBS, briefly dried to air, and fixed with 3% PFA. Cells were then blocked with 1% BSA-DPBS containing 0.5% Triton X-100. Cells were then incubated with the TUNEL reaction mix containing fluorescent-tagged dUTP for 2 h at room temperature. Cells were washed with DPBS and evaluated with a fluorescent microscope. Experiments were performed in duplicate and repeated at least three times. To quantitate an apoptosis index, the percentage of TUNEL-positive cells from six to eight midpower fields of each well were counted.

Rabbit carotid artery injury. After being sedated with subcutaneous ketamine (40 mg/kg) and xylazine (2 mg/kg), rabbits were intubated and anesthetized with 1% inhaled isofluorane. The right common carotid artery was injured by inflating a 3.0-mm angioplasty balloon twice to 10 atm for 30 s in an overlapping pattern, so that the entire vessel from aorta to carotid bifurcation was injured. The animals were euthanized 28 days after the initial procedure, the carotid arteries were collected and snap-frozen in optimum cutting temperature compound (Tissue-Tek, Miles; Elkhart, IN), and 8-µm-thick sections were obtained with a cryostat. Slides were blocked in 0.3% H₂O₂ in methanol for 30 min, incubated for 60 min at room temperature with a monoclonal anti-human Del1 antibody (1:300) in a humidified chamber, and then washed and incubated with biotin-labeled goat anti-rabbit polyclonal antiserum at room temperature for 30 min. Streptavidin-horseradish peroxidase (1:100) was added and incubated for 30 min at room temperature. Peroxidase color reaction was developed with 3,3'-diaminobenzidine (DAB) (Sigma). Five animals were used in these experiments, and multiple sections were evaluated from both vessels of each of three rabbits in detail. Representative sections are shown below.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Expression of recombinant Del1 proteins in Baculovirus. Wild-type Del1 isoforms were expressed in Sf9 insect cells and purified on a nickel column as previously described (15). Fractions were analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting with polyclonal Del1 antisera and were evaluated for endothelial cell attachment and angiogenic activity in the in ovo chick chorioallantoic membrane assay (data not shown).

Adhesion of VSMC to Del1. The adhesion of VSMC to insoluble Del1 was concentration dependent. Under SF conditions, the maximum amount of binding occurred when the coating concentration of Del1 was >10-20 nM (Fig. 1A). This binding was independent of active protein expression, as there was no difference in binding pattern in the presence or absence of 25 µg/ml cycloheximide (data not shown). VSMC bound to Del1-coated wells at the same rate as they bound to VN-coated wells (Fig. 1B). In these studies, 60% of total binding of cells to both Del1 and VN occurred by 1 h in culture, and the highest level of binding was reached at 3 h with no significant difference in the presence or absence of cycloheximide.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Vascular smooth muscle cells (VSMC) adhere to Del1. A: dose-dependent increases in adhesion of VSMC to Del1-coated wells. B: linear increase in the number of bound cells to 50 nM Del1. The rate of cell adhesion to Del1- and vitronectin (VN)-coated wells was similar. BSA, bovine serum albumin.

Adhesion of VSMC to Del1 requires integrin receptors. VSMC binding to Del1-coated wells was inhibited by pretreatment and subsequent culturing in the presence of RGD peptides (Fig. 2). This inhibition was dose related, with >80% inhibition of maximal binding in the presence of 100 µM RGD synthetic peptides. PCN-RGD peptides that are relatively specific for VN receptors inhibited adhesion with similar efficacy as the RGD peptides. There was no significant inhibition in the presence of nonspecific RGE control peptides. Inhibition of attachment in the presence of 100 µM peptides was similar for cells on Del1 and VN (Fig. 2B). Also, blocking antibodies to _v3 equally inhibited the binding of the VSMC to Del1 and VN. Antibodies to ₁ decreased the binding by 25%; however, antibodies to ₁-₃, ₅, and ₆ failed to have any significant inhibitory effect (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   VSMC adhesion to Del1 is mediated through VN receptors. A: concentration-dependent inhibition of adhesion of VSMC to Del1-coated wells by pretreatment with arginine-glycine-aspartic acid (RGD) peptides, with 80-90% inhibition occurring at 100 µM. The VN receptor-specific penicillamine (PCN)-containing peptide (PCN-RGD) was equally effective as the RGD peptides at the same concentration. There was <20% inhibition in the presence of equimolar GRGESP (RGE) control peptides. B: cell attachment to Del1 and VN was similarly blocked by incubation with RGD and PCN-RGD peptides at 100 µM concentration and a blocking antibody specific for the v3-integrin receptor at 50 µg/ml. Antibodies to 1-3-, 5-, and 6-integrin subunits inhibited adhesion <20%, and there was no significant inhibition in the presence of nonimmune serum (data not shown).

Chemotactic/migratory effects of Del1 on VSMC. The migratory effect of Del1 on VSMC was examined in a modified Boyden chamber (Fig. 3A). Placement of 20 nM Del1 or VN in the lower chamber induced the same amount of migration. When Del1 was placed in the upper chamber with the cells, there was minimal migration, similar to what has been observed with endothelial cells (15). Placing Del1 on top and VN in the bottom chamber compared the relative activity of VN and Del1. In these studies, Del1 competed with VN and inhibited the migratory response of VSMC to VN (data not shown). To demonstrate involvement of _v3 in the migratory response to Del1, VSMC were pretreated and cultured in the presence of blocking antibodies to _v3 or PCN-RGD peptides (Fig. 3B). _v3 antibodies inhibited the response to Del1 by ~50% and there was no significant inhibition in the presence of control antiserum (Fig. 3B). The PCN-RGD peptides inhibited 55-60% of the migratory response to Del1 and a similar effect was obtained with VN.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Del1 induces VSMC migration. A: in a modified Boyden chamber, equimolar amounts of Del1 or VN (50-100 nM) were placed either in the top chamber or the bottom chamber. After 6-8 h, the cells that had migrated to the bottom chamber were counted. Del1 and VN comparably stimulated VSMC migration when placed in the bottom chamber. This migration was not observed when the substrates were placed in the top chamber or BSA was placed in the bottom chamber as control (BSA). B: migratory response of VSMC to Del1 was mediated, at least in part, through v3. With pretreatment of cells and the addition of VN receptor-specific peptide antagonists (PCN-RGD) to the top chamber, there was ~50% decrease in Del1 and VN-stimulated migration. This inhibition was not seen with the negative control (RGE) peptides. Addition of blocking antibodies to v3 to the top chamber decreased migration of VSMC by ~50%. There was <10% inhibition of migration when nonimmune serum (NS) was added to the top chamber.

Focal contact assembly on Del1. VSMC adhered and spread on Del1-coated wells and developed the same pattern of stress fibers and focal contact assembly as cells on VN (Fig. 4). This was demonstrated by labeling with antibodies against smooth muscle actin and vinculin. Antibodies to both _v and ₃ localized these integrin subunits to the focal contacts, suggesting that VSMC adhesion to Del1 resulted in clustering of _v3-VN receptors to these sites (Fig. 4). Furthermore, this response to Del1 was not dependent on active protein synthesis, because there was no difference when these studies were performed in the presence of cycloheximide (data not shown). The clustering of integrins on Del1 was associated with phosphorylation of proteins within the adhesion focal contacts, as demonstrated by labeling of these sites with antiphosphotyrosine antibodies (data not shown).

View larger version (69K): [in this window] [in a new window]   Fig. 4.   VSMC on Del1 organize focal adhesion contacts that contain v3-integrin. VSMC were shown to adhere and spread similarly on culture wells coated with Del1 and VN. Soon after spreading, VSMC recruited VN and v3 to focal adhesion contacts. A similar pattern of stress fiber formation appeared on Del1 and VN, as seen here with smooth muscle -actin staining. Photos are magnified ×63.

Del1 supports growth of VSMC in SF conditions. When VSMC were cultured on Del1-coated wells, they were able to adhere, spread, and proliferate under SF conditions (Fig. 5). When plated on Del1, VSMC grew with a doubling time of 30-36 h, which was comparable to their growth rate in the presence of 10% FBS. When confluency was reached, cells plated on Del1 maintained a viable monolayer after 72 h in culture. Cells that were cultured on VN-coated wells were also able to adhere and spread under SF conditions and survive up to 48 h; however, there was limited growth of these cultures. By 48 h, cells on VN began to lose substrate adhesion and roundup.

View larger version (144K): [in this window] [in a new window]   Fig. 5.   Del1 supports the growth of VSMC in culture. VSMC were able to attach and spread on VN and Del1 under serum-free conditions. At 24 h, the cell number and phenotype were similar in Del1 and VN cultures. After 48 h, Del1 cultures were semiconfluent and similar to cultures containing serum in the media. However, at this time, there was minimal growth of the VN cultures and there were many floating and dead cells. By 72 h, the Del1 and serum-containing cultures were confluent and maintained their typical phenotype, whereas the VN cultures remained subconfluent. Photos are magnified ×10.

View larger version (31K): [in this window] [in a new window]   Fig. 6.   Del1 supports proliferation of VSMC in culture. A: VSMC on Del1 substrate were able to grow under serum-free conditions. The proliferation rate was dependent on the concentration of Del1 coating, with a plateau >50 nM of Del1. At each time point, the number of cells was quantitated with the use of a metabolic assay, as described in MATERIALS AND METHODS. B: proliferation of VSMC on 100 nM Del1 and VN was evaluated and compared over a 72-h period. There were significantly more cells in the Del1 cultures after 48 h, and this difference was maintained for 72 h. There was no cell survival on BSA-coated wells (data not shown). Each experimental time point was performed in triplicate, and the data points represent an average of three different experiments. C: VSMC expansion on Del1 under serum-free conditions was due in part to an inhibition of apoptosis. To determine the contribution of cell death to the difference observed between the VN and Del1 cultures, the percentage of TdT-mediated dUTP nick-end labeling (TUNEL)-positive cells was determined over a 72-h time course. The data points represent the ratio of TUNEL-positive cells to the total number of cells counted in 4-6 midpower fields. Each experiment was performed in duplicates and the data points represent an average of three different experiments. A similar percent of cells in Del1 and 10% fetal bovine serum (FBS) (supplemented) cultures underwent apoptosis at every time point. However, there was a progressive increase in the number of apoptotic VSMC on VN. D: VSMC expansion on Del1 was also due in part to increased cell division. Proliferating cell nunclear antigen (PCNA) labeling was performed and cells quantitated, with cells cultured in 10% FBS representing a positive control. Percent PCNA-positive cells represent the ratio of positive cells to the total counted cells in a midpower field. Each experiment was performed in duplicate, and 6-8 fields were counted in each set. The results represent an average of three different experiments.

Del1 expression is upregulated in the injured vascular wall. In light of these findings, the expression of Del1 by embryonic endothelial cells strongly suggests a role for Del1 in embryonic vascular remodeling. To investigate whether Del1 is expressed in the injured vessel wall, and thus might also have a role in pathological remodeling, immunohistochemistry experiments were performed on tissues harvested from a rabbit carotid injury model. A monoclonal antibody was used to evaluate Del1 expression in frozen sections of the tissue. In areas that were injured, but where there was no intimal thickening, Del1 expression was identified in a few remaining regenerating endothelial cells and also in some VSMC (Fig. 7A). In the noninjured contralateral carotid vessel no Del1 expression was detected in either the endothelial or VSMC (Fig. 7B). In vessels where significant intimal thickening was noted, Del1 expression was observed in multiple VSMC layers in the neointima and perhaps also in the media (Fig. 7C).

View larger version (120K): [in this window] [in a new window]   Fig. 7.   Del1 expression is reactivated in the setting of vascular injury. In tissues harvested from a rabbit carotid injury model, a monoclonal antibody was used to evaluate Del1 expression (dark brown staining) in frozen sections of tissue. A: in areas that were injured but there was no intimal thickening, Del1 expression was primarily restricted to the endothelial cell layer. B: control contralateral noninjured vessel showed no evidence of Del1 expression. C: in vessels where intimal thickening was noted, Del1 expression was also found in multiple cell layers under the endothelium. D: there was no labeling when the primary Del1 antibody was omitted. A and B are magnified ×4 and C and D at ×10.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Del1 has an endothelial cell-restricted and temporal pattern of expression during embryogenesis, suggesting a regulatory function for Del1 in vascular formation (6). It is now known that Del1 can interact with endothelial cells through the VN-integrin receptor _v3 (15). As described with other VN receptor substrates, Del1 signaling can mediate adhesion and migration of endothelial cells. In addition, Del1 has been shown to be proangiogenic in the chorioallantoic membrane assay, and this activity is novel among the VN-receptor- binding substrates. Del1 is thus likely to play an important role in modulating endothelial cell functions associated with vascular formation. The studies presented here suggest that Del1 may also activate paracrine signaling pathways that regulate VSMC migration and proliferation, extending the roles of Del1 to vessel wall development and remodeling. In most assays evaluated here, such as cell adhesion, migration, and focal contact formation, Del1 was functionally similar to ligands such as VN and thrombospondin, which can bind the VN receptor on endothelial and VSMC.

These studies show that Del1 supports VSMC expansion under SF conditions in vitro and that this effect is mediated in part through a decrease in programmed cell death. For instance, at 72 h, VSMC on Del1 maintained a healthy confluent monolayer, despite a low level of cell division. VSMC on VN were rapidly deteriorating at this time point. The protective effect of Del1 was demonstrated with TUNEL assays, which indicated a decrease in the overall level of apoptosis. These findings are in keeping with experiments demonstrating that Del1 supports proliferation of endothelial cells through inhibition of apoptosis (K. Penta and T. Quertermous, unpublished observations). Because activation of _v3 by other ligands has been shown to inhibit apoptosis of VSMC, it is likely that this interaction is responsible for the antiapoptotic actions of Del1 (7).

Perhaps the most remarkable finding of this study is the observation that Del1 is superior to VN for supporting VSMC growth under SF conditions. This Del1 function was concentration dependent, requiring 50-100 nM Del1 compared with 10-20 nM for adhesion and migration. VSMC grew at the same rate whether they were plated on Del1 matrix or grown in the presence of serum. However, there was a significant difference in growth rate between cells grown on Del1 versus VN. This difference was noted after 36-48 h, when cells on VN stopped dividing and subsequently did not reach confluency. In contrast, cultures grown on Del1 or in the presence of serum continued to divide and developed stable confluent monolayers within 72 h. This observation was supported by results demonstrating a fivefold increase in PCNA-positive cells on Del1 compared with VN.

There are several possible explanations for this observed increase in proliferation rate of VSMC plated on Del1 in the absence of exogenous growth factors. First, it is possible that the Del1 protein preparations used in these studies were contaminated with a growth factor that is active on these cells. This seems unlikely, given that the recombinant Del1 protein was made in insect cells, and multiple batches of protein were employed in these studies. In addition, these same preparations did not have growth factor activity for cultured endothelial cells. Second, it is possible that Del1 does not itself have growth factor activity but rather induces VSMC to produce a growth factor that functions in an autocrine fashion. For instance, VSMC are known to produce platelet-derived growth factor, and signaling activated by Del1 might initiate such expression. Third, the most intriguing possibility is that Del1 itself has intrinsic growth factor-like activity. This would not be surprising, because this molecule contains multiple EGF-like repeats that could function to activate signaling by a receptor tyrosine kinase or other form of growth factor receptor. Indeed, the angiogenic activity of Del1 protein argues for the presence of a growth-factor signaling activity in endothelial cells, because angiogenesis requires signaling through both growth factor and integrin-signaling pathways (5, 15). Whereas the overall organization of the two molecules is very different, there are a number of functional similarities between Del1 and thrombospondin, another RGD-containing matrix molecule that can prevent apoptosis and stimulate division of VSMC (14).

Del1 is thus likely to have both autocrine and paracrine effects in vascular development. The autocrine functions would serve to initiate and support angiogenesis, and the paracrine effects facilitate the migration and proliferation of VSMC. Del1 may thus have a broad role in embryonic vascular development and might serve as a particularly robust factor for promoting therapeutic angiogenesis in the setting of human vascular disease.

Equally interesting and important is the potential role for this molecule in vascular remodeling associated with vascular injury. The studies presented here show that Del1 expression is reactivated in the endothelium and vessel wall in the setting of vascular injury strongly suggest that this embryonic factor may have an important role in mediating aberrant vascular remodeling associated with vessel wall disease. It has been clearly established through experiments in animal models that _v3 has a critical role in mediating neointima formation and that blockade of this integrin limits lumen stenosis (3, 17). Osteopontin, thrombospondin, and other _v3-integrin ligands have been extensively studied in the setting of vessel injury in various animal models; however, the precise molecular species involved in human disease remains unclear. Del1 joins the list of molecules that may have a functional role in the vascular remodeling process that accompanies vessel injury.