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Endocrinology Vol. 140, No. 4 1575-1580
Copyright © 1999 by The Endocrine Society

ARTICLES

Connective Tissue Growth Factor (IGFBP-rP2) Expression and Regulation in Cultured Bovine Endothelial Cells1

Mary Boes, Brian L. Dake, Barbara A. Booth, Ngozi E. Erondu, Youngman Oh, Vivian Hwa, Ron Rosenfeld and Robert S. Bar

Department of Internal Medicine, Diabetes and Endocrinology Research Center, Veterans Administration Medical Center, The University of Iowa, Iowa City, Iowa 52246; and the Department of Pediatrics, Oregon Health Sciences University, Portland, Oregon 97201

Address all correspondence and requests for reprints to: Robert S. Bar, The University of Iowa, Department of Internal Medicine, ENDO-3E19 VA Medical Center, Iowa City, Iowa 52246. E-mail: rbar{at}icva.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Media from large vessel endothelial cells (pulmonary artery, aorta) contained intact connective tissue growth factor (CTGF) and a dominant 19-kDa band. N-terminal analysis of the 19-kDa band showed sequence corresponding to CTGF amino acid 181–190, suggesting that the 19-kDa band represented a proteolytic fragment of CTGF. Intact CTGF was increased by cAMP but not by transforming growth factor-ß (TGFß). CTGF messenger RNA (mRNA) was not changed by cAMP nor TGFß. In two microvessel endothelial cells, mRNA was found at low levels by PCR and Northern analysis, but no CTGF protein was seen on Western analysis. In the microvessel cells, TGFß increased and cAMP did not change CTGF mRNA levels, with neither TGFß nor cAMP increasing CTGF protein. The discordance between protein and mRNA levels in large vessel and microvessel endothelial cells was mostly explained by the effects of cAMP and TGFß on media proteolytic activity; in large vessel cells, cAMP inhibited degradation of CTGF, whereas in microvessel cells, TGFß and cAMP stimulated proteolytic activity against CTGF.

We conclude that in large vessel endothelial cells, cAMP increased intact CTGF protein by inhibiting degradation of CTGF, whereas TGFß stimulated neither CTGF mRNA nor protein; in microvessel cells, TGFß increased CTGF mRNA, while both TGFß and cAMP stimulated CTGF degradation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
KIM ET AL. (1) recently described a family of low affinity insulin-like growth factor binding proteins (IGFBPs), which are now referred to as IGFBP-rP1, -rP2, -rP3, and -rP4. Each represented a previously described complementary DNA (cDNA) that predicted a protein with structural similarity to the six known IGFBPs, IGFBP-1, through IGFBP-6. The four members of the low affinity IGFBPs are Mac25 (IGFBP-rP1), connective tissue growth factor (CTGF) (IGFBP-rP2), nov oncogene (IGFBP-rP3), and cyr 61 (IGFPB-rP4). These proteins have 30–40% structural homology to the six IGFBPs in the N-terminal domain, share at least 11 of the cysteines in this domain, and contain the N-terminal domain sequence GCGCCXXC (1, 2, 3, 4). Each binds IGF-I and IGF-II but with substantially lower affinity than the six known IGFBPs (1). CTGF (IGFBP-rP2) and cyr 61 (IGFBP-rP4) are products of immediate-early genes that are induced by specific growth factors or selected oncogenes.

CTGF or IGFBP-rP2 (4) was initially described by several groups in 1991. Bradham and Grotendorst (5) purified CTGF from medium conditioned by human umbilical vein endothelial (HUVE) cells, Ryseck et al. (6) described CTGF in mouse 3T3 cells and Brunner et al. (7) reported CTGF in TGFß-stimulated mouse AKR-2ß cells. The cDNA for CTGF was cloned by Grotendorst’s lab from an HUVE cDNA expression library (5). CTGF was shown to be a 38 kDa, 349 amino acid, 38 cysteine-rich protein, with 22 cysteines clustered in the N-terminal domain, and the other 16 cysteines aggregated in the C-terminal region. As in the other two members of this family, CTGF is composed of four modules, an N-terminal IGF binding domain, a von Willebrand factor type C repeat domain, a thrombospondin type 1 repeat module and a C-terminal domain homologous to the Drosophila slit protein (3). Despite having little sequence homology to platelet-derived growth factor (PDGF), CTGF is antigenically and functionally related to PDGF, being recognized by anti-PDGF antibodies, having overlapping chemotactic and mitogenic activities with PDGF and accounting for greater than 90% of the bioactivity (chemotaxis and mitogenesis of NIH 3T3 cells) in HUVE conditioned media that had previously been attributed to PDGF (5).

The precise functions of CTGF are unknown. However, indirect evidence suggests a role for CTGF in atherosclerosis (8), wound healing (9, 10), and as a downstream regulator of TGFß action (9, 10, 11). CTGF messenger RNA (mRNA) is highly expressed in endothelial and smooth muscle cells of atherosclerotic blood vessels, but not in homologous nonatherosclerotic normal vessels (8, 12). In human skin fibroblasts, transforming growth factor-ß (TGFß) promotes wound healing with initially elevated levels of TGFß followed by increased CTGF (10); growth factors other than TGFß, including epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and PDGF, did not activate CTGF (9). The CTGF gene has a novel TGFß response element in its promoter (11), apparently accounting for the universal stimulation of CTGF by TGFß in all cells producing CTGF. Because CTGF was first described in conditioned media of endothelial cells, in the present study we have evaluated the expression and regulation of CTGF (IGFBP-rP2) in cultured endothelial cells derived from bovine blood vessels and, to a lesser extent, endothelial cells cultured from human vessels.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Bovine endothelial cells from pulmonary artery, aorta, omental fat, and periaortic fat were prepared and characterized as previously described (13). Media was concentrated in a centricon-10 to approximately 35x (Amicon, Inc., Beverly, MA). The concentrated media was used for Western analyses and degradation studies. Human endothelial cells from pulmonary artery (HPAEC) and pulmonary microvessels were purchased from Clonetics (Walkersville, MD). For experiments with HPAEC, cells were grown to confluence in medium EGM-2 (Clonetics) then weaned from EGM-2 medium by changing medium to 50% EGM-2 and 50% EBM-2 (Clonetics) for 24 h then 25% EGM-2 and 75% EBM-2 for 24 h then 100% EBM-2 plus 10 ng/ml EGF. For 18 h experiments, media and cells were then processed as described for the bovine cells.

Materials
cAMP was purchased from Sigma Chemical Co. (St. Louis, MO), TGFß1, 2, from R&D Systems (Minneapolis, MN), and forskolin from Calbiochem (San Diego, CA).

NH2-terminal amino acid sequencing was performed from protein bands blotted onto polyvinylidene difluoride (Pro-Blott; PE Applied Biosystems, Forest City, CA) using a phenylthiohydantoin analyzer as previously described (14).

RNA isolation and Northern blot analysis (13)
Total RNA was isolated from cells using RNeasy Mini Kit, Qiagen (Santa Clarita, CA). Total RNA was quantitated by absorbance at 260 nm. For Northern analysis, 5 µg total RNA was applied to each lane. The accuracy of quantitation was verified by size-separating the RNA on a denaturing agarose/formaldehyde gel and comparing the intensities of 28 S and 18 S ribosomal RNA bands by ethidium bromide staining. The RNA was transferred to a nylon membrane (Gene Screen, New England Nuclear, Boston, MA) using a Hoefer Transphor Model TE52 transfer apparatus (Hoefer Scientific, San Francisco, CA) and fixed to the membrane using a Stratalinker UV cross-linker (Stratagene, La Jolla, CA) with exposure of 120,000 microjoules/cm2. Prehybridizations were performed under high stringency conditions in a sealed pouch in 5 x SSPE (1 x SSPE = 0.18 M NaCl, 10 mM NaH2PO4, 1 mM EDTA, pH 7.4), 50% formamide, 10% dextran sulfate, 10 x Denhardt’s solution, 1% SDS, and 100 µg/ml denatured salmon sperm DNA for 2 h at 50 C. The filters were hybridized with a CTGF cDNA that was labeled with [32P] dCTP by random priming. After heat denaturing (10 min at 100 C) the 32P-labeled probe (1 x 106 cpm/ml/lane) was added and hybridization continued for 18 h at 50 C. The filters were washed twice with 2 x SSPE plus 0.2% SDS for 30 min each at 60 C, then twice with 0.1 x SSPE for 15 min at 60 C, and finally exposed to Kodak X-Omat AR film for autoradiography.

Western analysis electrotransfer
Protein blotting was performed for 90 min at 4 C using a Hoefer Transphor Model TE52 at 0.8 A in a buffer consisting of 25 mM Tris-base, 194 mM glycine, and 20% methanol. After transfer, the membrane was dried at 37 C and blocked according to the method of Yang et al. (15). Blots were blocked in 4% nonfat milk made up in TBS-T (20 mM Tris base, 137 mM NaCl, 0.1% Tween 20, pH 7.6) for 1 h at room temperature. The membrane was rinsed with TBS-T then primary antibody (1:3000) in 4% milk/TBS-T was added for 1 h at room temperature and incubated at 4 C for 18 h. The membrane was next washed x4 with TBS-T, the first wash with 15 min exposure, and the next three washes for 5 min each. The second antibody (antirabbit IgG-horse radish peroxidase conjugate) in TBS-T (1:3000 dilution) was next added, allowed to incubate for 1 h, then the membrane was washed four times and incubated with chemiluminescence reagents (Western Blot Chemiluminescence Reagent (Dupont NEN, Boston, MA). The membrane was blotted on Whatman paper, sealed in Seal-a-Meal, then exposed to film for 2–20 min.

The CTGF antiserum was prepared from serum of rabbits injected with recombinant human IGFBP-rP2 (15). It does not cross-react with IGFBP-1 through IGFBP-6, IGFBP-rP1, or PDGF BB and does cross-react with bovine CTGF (15).

Degradation of 125I-CTGF
CTGF produced in baculovirus (1, 15) was iodinated with 125I using the lactoperoxidase method and purified on a Sephadex G100/40–120 (Pharmacia, Piscataway, NJ) column (0.9 x 55 cm). Conditioned media from cells exposed to medium alone and cells exposed to cAMP (10 mM) or TGFß1 (1 nM) were incubated with 125I-CTGF (~25,000 cpm) in a total volume of 25 µl for 18 h at 37 C, then stopped by adding 1 µl 25 mM PMSF, followed by 26 µl 2 x SDS-PAGE sample buffer and applied to 16% SDS-PAGE and the dried gel exposed to Kodak X-O mat AR film overnight.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Bovine endothelial cells
CTGF (IGFBP-rP2) immunoreactivity was found in endothelial cells cultured from bovine pulmonary artery and aorta (Fig. 1Go). In conditioned media from both bovine aorta and pulmonary artery cells a doublet was observed at approximately 31 and 33 kDa, with the most intense immunoreactive band at approximately 19 kDa (Fig. 1Go, top panel). The 19-kDa band contained two peptides. The major peptide had the N-terminal sequence AYRPEDTFGP which corresponds to 9 of 10 amino acids of bovine CTGF beginning at amino acid 181, with the published CTGF primary sequence being AYRLEDTFGP. If the 19-kDa peptide contained the complete C-terminal portion of CTGF, its estimated size would be approximately 20 kDa, which closely approximates the 19-kDa band observed in Fig. 1Go.



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  		Figure 1. Regulation of CTGF by cAMP (1, 10 mM), TGFß1 (0.1, 1.0 nM) and forskolin (30 µM) in bovine pulmonary artery endothelial cells (left panel) and cAMP (10 mM) and TGFß1 (1.0 nM) in bovine aorta (right panel). Western ligand blots are in top panels and Northern blots in the lower panels.

 
Regulation of CTGF in large vessel bovine endothelial cells
Regulation of CTGF by TGFß, cAMP, and forskolin was assessed in the bovine large vessel endothelial cell cultures derived from the pulmonary artery (bPA) and aorta (bAo). In five experiments with bPA cells and two experiments with bAo cells, TGFß1 (1 nM) had no effect on CTGF protein or mRNA (Fig. 1Go). TGFß2 (n = 2 for bPA, n = 1 for bAo) also had no effect on CTGF mRNA or protein levels. Treatment with cAMP and forskolin increased intact CTGF (Fig. 1Go). Stimulatory effects were observed at 0.1 mM cAMP with progressively increased immunoreactivity in the 31- and 33-kDa bands up to 10 mM cAMP, the highest concentration of cAMP tested (Fig. 2Go). The increases in immunoreactivity were observed in both the 31- and 33-kDa bands without obvious change in the 19-kDa band. However, because of the intensity of the 19-kDa band, a decrease or increase of 10–15% might not be detectable. In seven experiments with bPA and two experiments with bAo, cAMP caused little change in CTGF mRNA levels (Fig. 1Go, bottom panel), despite the increase in CTGF protein.



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  		Figure 2. Dose response effect of cAMP on CTGF protein (top panel) and mRNA (lower panel) in bovine pulmonary artery endothelial cells. Cells were exposed to cAMP (0.01, 0.1, 1, and 10 mM) for 18 h.

 
Regulation of CTGF in bovine microvessel endothelial cells
Two bovine microvessel endothelial cell cultures, derived from periaortic (bPM) and omental fat (bOM) were evaluated. The bPM cells showed no detectable CTGF protein and significantly lower levels (compared with bPA and bAo cell) of CTGF mRNA after 30-min exposure of Northern blots (n = 2), conditions used in the evaluation of the pulmonary artery and aorta endothelial cells (Figs. 1Go and 2Go). To be certain that CTGF mRNA was present in the microvessel cells, RT-PCR of microvessel RNA was performed, using primers designed to homologous regions of human and mouse CTGF mRNA. These primers had previously been shown to amplify bands of appropriate size and sequence in the bovine large vessel endothelial cells. When used to amplify microvessel cDNA, both the periaortic and omental microvessel RNA showed a band of appropriate size and sequence,2 indicating that low levels of CTGF mRNA were present in the microvessel cells.

When the bPM cells were exposed to cAMP, which increased expression of CTGF in pulmonary artery and aorta endothelial cells, there was little effect on CTGF protein. With longer exposure (18 h) of Northern blots, cAMP caused little change in mRNA in bOM cells (Fig. 3Go, left) while decreasing mRNA in bPM cells (Fig. 3Go, right). TGFß1 (1 nM) had no effect on CTGF protein but did increase CTGF mRNA in both types of microvessel cells (Fig. 3Go, bottom panels).



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  		Figure 3. The effect of cAMP (10 mM) and TGFß1 (1 nM) on CTGF protein (top panel) and mRNA (bottom panel) in bovine microvessel endothelial cells. Cells from omental fat (bOM) are in the left panel and from periaortic fat in the right panel.

 
Human pulmonary artery endothelial cells
Western blots of media conditioned by human pulmonary artery cells were different than analogous studies with bovine pulmonary artery and aorta cells (Figs. 1Go and 2Go). The immunoreactive CTGF showed three bands in the 35-kDa region vs. 2 bands in the bovine cells, the human CTGF being 3–4 kDa larger than bovine CTGF. Three bands of CTGF immunoreactivity were also observed in human microvessel pulmonary endothelial cells (data not shown) and in human recombinant CTGF (Fig. 4Go). Treatment of the human media with N-glycanase changed the Western blot to two bands at approximately 31 and 33 kDa, similar to the bovine cells. Thus, the differences in CTGF between the bovine and human cells likely represent different glycosylation of the CTGF protein. Furthermore, in the human cells there was a minimally detectable 19-kDa immunoreactive band compared with the intense staining of the 19-kDa band seen in the bovine cells (Fig. 4Go).



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  		Figure 4. Western immunoblot of conditioned medium from human pulmonary artery endothelial cells (hPA CM, lane 2) compared with conditioned medium from bovine pulmonary artery cells (bPA CM, lane 1), recombinant human CTGF (rh CTGF, lane 3) and 125I-labeled hCTGF (125I-CTGF, lane 4).

 
Effect of cAMP on the degradation of 125I-hCTGF and on cell associated CTGF in bovine PA and aorta cells
CTGF degradation
To potentially explain the discordant effects of cAMP on CTGF in the bovine large vessel cells, i.e. increased intact CTGF without a concomitant increase in mRNA, two alternative mechanisms were studied: cAMP effect on CTGF degradation and cAMP alteration of cell-associated vs. soluble CTGF. These two processes were selected for further study because of the substantial proteolytic activity against CTGF in the large vessel bovine cell cultures and because intact CTGF has recently been shown to be preferentially cell-associated in human fibroblasts whereas smaller fragment(s) of CTGF were preferentially found in the media (16).

For proteolysis studies, baculovirus produced recombinant human CTGF (Fig. 4Go) was iodinated and purified by gel filtration.3 Endothelial cells were incubated with and without cAMP (10 µM). After 18 h at 37 C, conditions identical to those used in the CTGF regulation in Figs. 1–3GoGoGo, the media was collected and concentrated. The concentrated media was incubated with 125I-CTGF for 18 h, run on SDS-PAGE and autoradiograms developed (Fig. 5Go). In bAo media, cAMP decreased the degradation of 125I-CTGF (Fig. 5Go, bAo, n = 3), with the density of intact 125I-CTGF being approximately 180% of control. The results of several experiments with bPA cells were inconsistent. For bPA, the majority of the intact 125I-CTGF was degraded in control cells with three bands observed at approximately 25, 23, and 17 kDa (Fig. 5Go, control).



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  		Figure 5. Degradation of 125I-hCTGF by conditioned media from bovine pulmonary artery cells treated with cAMP (10 mM panel 1 n = 3, panel 2 n = 2), bovine aortic endothelial cells (bAo, panel 3), bovine omental fat microvessels treated with cAMP (10 mM) or TGFß1 (1.0 nM) in panel 4, human pulmonary artery cells treated with cAMP (10 mM) in panel 5 and control 125I-CTGF. In each experiment, cells were incubated with control medium, cAMP or TGFß1 for 18 h, the conditioned media removed then exposed to 125I-CTGF (25,000 cpm) for 18 h before being run on 12% SDS-PAGE.

 
In three experiments, cAMP inhibited the degradation of 125I-CTGF resulting in increased 37-kDa bands, a potentially viable explanation for the discordant effect of cAMP on CTGF protein and mRNA levels (Fig. 5Go, bPA, first panel). However, in other experiments (n = 2) cAMP had less effect on CTGF degradation (Fig. 5Go, bPA, second panel). In hPA cells, basal degradation was much lower than in bPA, with the majority of the 125I-CTGF remaining in the 37-kDa band (Fig. 5Go, hPA); cAMP had little effect on the proteolysis of CTGF in the human PA cells. In bOM cells, basal (control) degradation was less than that observed in bPA (Fig. 5Go, bOM); in the presence of cAMP, or TGFß, rates of proteolysis of the intact 37 kDa CTGF were increased, with loss of the 37-kDa band and increased prominence of bands at 23 and 17 kDa (Fig. 5Go, bOM).

Thus, in bOM, intact CTGF was not observed in cells exposed to cAMP or TGFß1 despite the finding that TGFß clearly stimulated CTGF mRNA (Fig. 3Go) because both cAMP and TGFß increased the proteolysis of intact CTGF. For the large vessel bAo, cAMP decreased degradation of CTGF, which could explain the finding of increased CTGF without an increase in steady-state mRNA. Results with bPA were too inconsistent to definitively ascribe a similar mechanism to account for the increased CTGF protein without concordant increases in CTGF mRNA.

Cell-associated CTGF
When bPA cells that had been treated with cAMP were lysed with 1 x SDS-PAGE sample buffer minimal intact or degraded CTGF was solubilized (data not shown). Furthermore, there was no obvious difference in cell-associated CTGF or CTGF fragments in control cells or cells treated with cAMP.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Connective tissue growth factor (CTGF or IGFBP-rP2) was initially described by Bradham and Grotendorst in a human umbilical vein endothelial cell (HUVEC) cDNA expression library (5). Despite having little structural homology to PDGF, CTGF was detected by its reactivity with PDGF antibodies and was subsequently demonstrated to account for greater than 90% of the bioactivity in HUVEC conditioned medium previously attributed to PDGF. Because human CTGF mRNA has been reported to be present in multiple tissues throughout the body (12) and the initial report of CTGF mRNA came from studies with umbilical vein endothelial cells, it is perhaps not surprising that other cultured endothelial cells, such as the bovine and human endothelial cells of this study, would also produce CTGF. However, several findings of the present report would not have been predicted by the existing literature. First, TGFß did not stimulate CTGF mRNA or protein in the bovine and human large vessel endothelial cells, despite the finding that TGFß1 has been previously reported to stimulate CTGF in all other cultured cells that express the protein (10, 11, 12). In fibroblasts, Grotendorst (11) has defined a novel TGFß response element in the CTGF promoter, in which point mutations resulted in complete loss of TGFß response. Because of the close association between TGFß and CTGF, one of the more commonly postulated functions of CTGF suggests that CTGF represents a downstream target for mediating selected TGFß actions (10, 11, 12). Second, cAMP and forskolin stimulated CTGF expression in the large vessel bovine endothelial cells. Other growth factors, including IGF-I, IGF-II, insulin, EGF, PDGF, and bFGF did not increase CTGF in the large vessel endothelial cells. No previous study has reported regulation of CTGF by cAMP or by agents which stimulate cAMP. Within the same bovine species, cAMP had differential effects on CTGF proteolysis. Both large vessel and microvessel bovine cells had substantial rates of basal proteolytic activity against CTGF. However, whereas cAMP inhibited CTGF proteolysis in bovine aorta cells, cAMP and TGFß1 increased proteolytic activity in the microvessel cells. Third, large vessel, but not microvessel, bovine endothelial cells, express substantial amounts of CTGF protein and mRNA. The two bovine microvessel endothelial cells reported in this study were derived from periaortic fat and omental fat. Both were shown to have CTGF mRNA by RT-PCR amplification and by Northern analysis but expressed intact CTGF protein at levels too low to be detected by standard Western immunoblot. Depending on the functions of CTGF, the differential expression, and perhaps differential regulation, of CTGF in microvessel vs. large vessel endothelial cells has obvious implications. Finally, the bovine, but not the human, endothelial cells expressed substantial proteolytic activity against CTGF. In the bovine pulmonary artery cells, the strongest immunoreactivity in Western blots was against a 19-kDa band, whose N-terminal amino acid sequence suggested that it encompassed the distal two thirds of the CTGF protein and was likely a proteolytic fragment of CTGF. The functions of this fragment, as well as the other proteolytic fragments of CTGF, are of potential interest, especially in light of a recent report by Brigstock et al. (17). These authors described a 10-kDa heparin-binding protein in pig uterine washings with N-terminal sequence corresponding to the distal third of porcine CTGF. This 10-kDa fragment stimulated DNA synthesis in fibroblasts and smooth muscle cells, but not in endothelial cells, suggesting that the 10-kDa fragment functioned as a selective effector of embryo growth signaling in the pig. Whether other larger, C-terminal fragments of CTGF, such as the 19-kDa peptide described in this report, could have specific mitogenic or chemotactic properties remains to be determined.

The precise functions of CTGF, in vitro and in vivo, are unknown. Although reflecting a rather modest literature, perhaps the three most commonly suggested functions of CTGF relate to its potential role in wound healing, its involvement in the development and progression of atherosclerosis and its ability to act as a downstream effector of TGFß action (10, 11, 12). The indirect data suggesting a role of CTGF in wound healing include the mitogenic and chemotactic properties of CTGF for cells involved in wound repair (9), the coordinate overexpression of CTGF and TGFß mRNA in a rat model of wound repair (10) and the presence of a specific response element in the CTGF promoter of fibroblasts for TGFß (11), a growth factor important in wound repair (18) and, perhaps, atherosclerosis (19). The involvement of CTGF in atherosclerosis is suggested by the finding that CTGF is expressed differentially in atherosclerotic vs. normal blood vessels (8). Endothelial and vascular smooth muscle cells of atherosclerotic blood vessels have substantially greater CTGF mRNA than comparable cells in normal, nonatherosclerotic vessels. Furthermore, TGFß1, which has been reported to stimulate CTGF in human VSMC, induces overproduction of extracellular matrix proteins in intimal vascular smooth muscle cells, suggesting that TGFß, via CTGF, may regulate extracellular matrix production in these cells, leading to the initimal thickening that characterizes atherosclerosis.

It will also be important to determine how CTGF, acting as a low affinity IGF binding protein, will influence IGF function in the vessel wall. Vascular smooth muscle cells produce IGF-I, IGFBPs (20, 21), IGFBP proteases (21, 22) and have specific surface receptors for the IGFs (23). Injury to the arterial wall induces increased IGF-I mRNA and protein in the region of injury. Furthermore, IGFs have been postulated to play a role in wound healing and atherosclerosis, two processes in which CTGF is thought involved. It will now be of particular interest to determine the interactions of the low affinity IGF binding protein CTGF (IGFBP-rP2) with the IGF system, especially as such interactions relate to wound healing and atherosclerosis.

Finally, there are several issues raised by this report that need to be addressed before the regulation and functions of CTGF can be better defined. These include, but are certainly not limited to, the following: 1) the lack of TGFß stimulation of CTGF in bPA and bAo cells; 2) an explanation for different CTGF expression in human vs. bovine cultured endothelial cells; 3) understanding the mechanisms of CTGF proteolysis and its regulation in endothelial cells; 4) characterization of the dominant endothelial protease(s) for CTGF; and 5) the precise functions of CTGF and its proteolytic fragments in cultured cells and in intact blood vessels. Answers to questions such as these should provide important insights into the roles of CTGF, a recently described and potentially important protein that may be a critical mediator of growth factor effects.


   Footnotes
 
1 This work was supported by funds from Veterans Affairs research, National Institutes of Health Grants DK-25421, DK-25295, DK-51513, CA58110, U.S. Army DAMD17–96-1–6204 and Diagnostics Systems Laboratories, Inc. (Webster, TX). Back

2 Amplified band = 285 bp with sequence identical to bovine CTGF. Back

3 For comparison to studies in the bovine cells ( Figs. 1–3GoGoGo), the recombinant human CTGF is approximately 4 kDa larger in SDS-PAGE than the bovine CTGF. Back

Received September 14, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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