Biochemical analysis of a human humoral fibroblast inhibitory factor associated with impaired vascular prosthetic graft incorporation

Biochemical analysis of a human humoral fibroblast inhibitory factor associated wi t impaired vascular prosthetic graft incorporation Jacob Schne]derman, MD, Sarah KnoUer, PhD, Raphael Adar, MD, and N a p h t a l i Savion, P h D , Tel Aviv, Israel A specific inhibitory factor was isolated from human serum o f a patient who manifested impaired prosthetic graft tissue incorporation. The later reaction was hypothesized to be related to intfibiting fibroblast proliferation by a specific humorai factor. The crude inhibitory senam was tested against a pool o f normal human sera with different cell types in culture. Proliferation o f human skin fibroblast and human smooth muscle cell cultures incubated in the presence o f 50% inhibitory serum were inhibited up to 58% and 37%, respectively. Proliferation o f bovine capillary endothelial cell cultures was stimulated under similar conditions. Isolation and purification o f the inhibitory factor from crude serum were initiated by ammonium sulfate precipitation. The pellet was further fractionated by sepharose 6B gel filtration. The inhibitory activity was eluted from the column in a relatively purified form as indicated by gel electrophoresis o f the inhibitory fractions, which demonstrated a specific band corresponding to the inhibitory protein, with an apparent relative molecular mass o f 230 kDa. The inhibitory factor showed a high affinity to concanavalin A, indicating its nature as a glycoprotein not associated with albumin or inlmunoglobulin fractions o f the serum. (J VAse S t ~ c 1991;14:103-10.)

Perigraft seroma is a rare complication encountered after implanting prosthetic vascular grafts, mainly in an extraanatomic position. 1The pathogenesis of this complication is not fully understood, and several theories have been suggested: (1) immunologic or allergic reaction to the implanted graft; 2 (2) serum transudation through the prosthetic graft; 3'* (3) plasma extravasation from perigraft capillaries into the surrounding conduit; ~ and (4) a humoral fibroblast proliferation inhibitory factor. 6 Despite present controversy concerning the exact origin of perigraft seroma, accumulating clinical and histologic data exist that support the assumption that an induced fibroblast irthibitory factor may lead to From the Department of General and Vascular Surgery, Sheba Medical Center (Drs. Schneiderman and Adar), and The Maurice and Gabriela Goldschleger Eye Research Institute, Sackler Faculty of Medicine (Drs. KnoUerand Savion), Td-Aviv University, Tel-Aviv. Supported by a grant from the National Council for Research and Development, Israel, and the Gesellschaft fiir Strahlen und Umwelfforschung GmbH, Mfinchen, Federal Republic of Germany. Reprint requests: Naphtali. Savion, MD, Goldschleger Eye Research Institute, Sheba Medical Center, Tel-Hashomer, 52621, Israel. 24/4/27971

defective incorporation of the artificial conduit into the tissue. The development of this humoral activity may be due to an allergic or foreign body reaction to the artificial graft implant. Sladen et al.6 were the first to propose the humoral factor theory, demonstrating the disappearance of perigraft seroma accumulation after plasmapheresis was performed on their patients. This could be attributed to filtering out of the humoral fibroblast inhibitory factor. The purpose of this study was to evaluate the proliferative properties of the inhibitory factor assumed to be within the serum of a patient with perigraft seroma, isolate the inhibitory fraction, and characterize it biochemically. CASE R E P O R T A 65-year-old woman was admitted for evaluation of a tender right groin mass that developed over a period of several weeks. Four years before she was admitted she underwent an aortobiprofunda bypass for disabling claudication (USCI ext. velour, 16:8 Dacron graft; U.S.C.I. Div. of C. R. Bard, BiUerica, Mass.). Two years after surgery the left limb of the graft was revised, after rupture of a femoral pseudoaneurysm. The distal portion of the conduit on the left was then replaced by a 6 mm polytetrafluoroethylene graft. On admission, a pulsatile 103

104 Schneiderman et al.

right groin mass measuring 4 x 4 cm was noted and suspected on clinical grounds to be a femoral pseudoaneurysm. Popliteal and pedal pulses were absent in both limbs. Ankle to arm indexes were 0.56 bilaterally. Ultrasonic evaluation demonstrated a large amount of fluid surrounding the right femoral graft. A femoral anastomotic pseudoaneurysm could not be ruled out; however, arteriographic findings did not support this diagnosis. The right groin was then explored surgically. The distal portion of the right limb of the graft was surrounded by clear serous fluid, well contained within a thick glistening capsule. The femoral graft and its anastomosis to the deep femoral artery were visualized and found to be intact. The fluid and capsule wall were cultured for bacteria and fungi and were found to be sterile. The patient had an uneventful recovery; however, 4 weeks later the right groin mass recurred. Throughout the next year the patient was asymptomatic and underwent surveillance by repeated ultrasonic examinations. The latter revealed no significant change in size and shape of the right femoral perigraft seroma, and no fluid was visualized surrounding the intraabdominal segment or left limb of the graft. Over the following 3 years the patient sustained three episodes of acute thrombosis of the graft, twice involving its left limb, and once involving the right limb. Those were treated successfully by Fogarty thrombectomy, intraarterial fibrinolysis with completion thrombectomy, and thrombectomy followed by polytetrafluoroethylene angioplasty of the deep femoral artery, respectively. After the first thrombotic event a thorough coagulation system evaluation was done. The latter did not reveal any obvious cause for the apparent hypercoagulability. Chronic warfarin (Coumadin) treatment, initiated after the first thrombotic episode, failed to prevent further events of graft thrombosis. The patient died of a cerebrovascular accident a few days after abdominal surgery for gangrenous acalculus cholecystitis. In our view this patient presented with the major complications attributable to a poorly incorporated vascular graft, namely, refractory perigraft seroma, anastomotic pseudoaneurysm, and tendency for graft thrombosis. MATERIAL AND METHODS Materials. Dulbecco's modified Eagle's medium (low glucose) (DMEM) was purchased from Grand Island Biological Co. (Grand Island, N.Y.). Fetal calf serum, newborn calf serum, penicillin, streptomycin, fungizone, glutamine, and trypsin versene solution were obtained from Biological Industries (Beth Haemek, Israel). Both sera were heat inactivated at 56 ° C for 30 minutes before use. Fibroblast growth factor was prepared as described. 7 Tissue culture dishes were purchased from Nunc (Roskilde, Denmark). Relative molecular mass (Mr) standards for gel electrophoresis (45-200 kd) were obtained from Bio-Rad (Richmond, Calif.) and for gel

journal of VASCULAR SURGERY

filtration (29-700 kd) from Sigma (St. Louis, Mo.). Sepharose 6B and CNBr-activated sepharose 4B were purchased from Pharmacia (Uppsala, Sweden). Concanavalin A and goat anti-(human serum albumin) were purchased from BioMakor (Rehovot, Israel). Goat anti-(human immunoglobulins) immunoglobulin G fraction were obtained from United States Biochemical Corp. (Cleveland, Ohio). (3- [{3-Cholamidopropyl}-dimethylammonio]-l-propanesulfonate) (CHAPS) was obtained from Sigma. Cells and culture conditions. H u m a n skin fibroblast (HSF) cells were prepared from skin biopsies taken from normal volunteers and from the patient who manifested impaired prosthetic graft tissue incorporation. H u m a n skin fibroblast cells were grown in culture at 37 ° C in 10% CO2 in D M E M containing 10% fetal calf serum, glutamine (2 mmol/L), streptomycin (100 ~g/ml), penicillin (100 units/ml) and fimgizone (0.25 t*g/ml). Cultures were passaged every 2 weeks, and passages 3 to 7 were used for experiments. H u m a n saphenous vein smooth muscle cells were prepared from tissue explants and grown as described for HSF, passaged every week, and passages 3 to 7 were used for experiments. Bovine brain capillary endothelial cells were prepared as described and grown as described for HSF cells, 8 but in the presence of 5% fetal calf serum, 5% newborn calf serum, and fibroblast growth factor (100 ng/ml) added to the cultures every other day until the cultures were closely confluent. To evaluate the effect of the various sera on cell proliferation, the original cultures were removed with trypsin and plated at an initial cell density of 25,000 ceUs/35 m m dish for fibroblast and smooth muscle cells, and 40,000 cells/35 mm dish for endothelial cells. The cultures were maintained in a regular growth medium for 24 hours then washed with DMEM and exposed to fresh growth medium containing the tested sera at concentrations of 10% to 50%. In the process of isolation and purification, various sera fractions were tested at concentrations of 5% to 25% in the presence of 25% pooled normal human sera (NHS). By day 6, when reaching subconfluence, the cultures were removed with trypsin and the cell number counted. Preparation o f serum. Blood samples were drawn and stored at room temperature for 1 hour, then overnight at 4 ° C, followed by centrifugation at 2000 rpm for 10 minutes. The serum was collected and stored at - 2 0 ° C. A m m o n i u m sulfate precipitation. Inhibitory serum (5 ml) and N H S (equivalent amount of

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july 1991

protein to the inhibitory serum) were mixed with an equal volume of 50% saturation ammonium sulfate (pH 7.0) and mixed at 4 ° C for 30 minutes followed by centrifugation at 10,000 rpm at 4 ° C for i0 minutes. The pellet was washed once with 25% saturation ammonium sulfate solution, then dissolved in water and extensively dialyzed against Dulbecco's modified phosphate-buffered saline (DPBS). The supernatant was concentrated by an Amicon (Amicon Div. of W. R. Grace & Co., Beverly, Mass.) cell concentrator by use of YM-10 membrane to a volume of 5 ml and then dialyzed against DPBS. Gel filtration. The ammonium sulfate dialyzed pellet containing 0.04% CHAPS, was applied to a sepharose 6B column (90 x 1.6 cm) and elutedwith DPBS containing 0.04% CHAPS at a rate of 20 ml/hour. Fractions (2.5 ml) were collected, and the A280 was measured for each fraction. Affinity chromatography. Albumin was separated from the active fractions and collected from the sepharose 6B column by the use of a goat anti(human serum albumin) sepharose column. The column was prepared by coupling the affinity purified antibodies against human serum albumin to CNBractivated sepharose. The column was equilibrated with DPBS, and the albumin fraction was eluted from the column by use of a MgCI2 (4.9 tool/L) solution followed by dialysis against DPBS. The albumin-free fraction was collected and a sample applied to goat anti-(human immunoglobulins) sepharose column. The column was prepared as described above for the other antibody. An albumin and immunoglobulin-free fraction was collected. The immunoglobulin fraction was eluted with MgCI2 (4.9 mol/L) sohation followed by dialysis against DPBS. Concanavalin A was coupled to CNBractivated sepharose, and the column was equilibrated with 20 mol/L Tris-HCl (pH 7.4), 0.5 mol/L NaCI and 5 mmol/L CaCI2 solution. Aliquot of the albumin and immunoglobulin-free fraction was applied to this column, and the bound material was eluted with 0.5 mmol/% a-methyl-D-mannoside and dialyzed against DPBS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein samples taken from the pooled concentrated fractions of the second sepharose column were dissolved in SDSsample buffer. Aliquots of 50 mm 3 were applied to polyacrylamide gel (7.5%) prepared according to the method of Laemmli.9 Protein bands were stained with Coomassie brilliant blue.

Fibroblast inhibitory factor and perigraft seroma

105

RESULTS Inhibition of cell proliferation by serum of

patient with perigraft seroma Exposure of skin fibroblasts derived from the patient with perigr~ft seroma (autologous fibroblasts) to the serum (numerous samples taken over a period of 6 months) ]prepared from the same patient (inhibitory serum), at a final concentration of 50%, resulted in a 58% inhibition of cell proliferation when compared to the same cells exposed to pooled NHS (Table I). The inhibitory activity of the serum derived from the patient with seroma was not limited to autologous fibroblasts. Homologous HSF cells (Two lines) were inhibited by the same concentration of that serum, but to a lesser degree (37%). Similar inhibition by that specific serum was observed in cultures of human smooth muscle cells. Exposure of bovine brain capillary endothelial cells to the inhibitory serum resulted in a significant stimulation of cell proliferation compared to cultures exposed to pooled NHS (Table I). The morphology of HSF and bovine brain capillary endothelial cells growing for 6 days in the presence of the inhibitory serum or pooled NHS is shown in Fig. 1. Human skin fibroblast cells exposed to both sera demonstrated normal fibroblastic morphology of spindle-like cells. The inhibitory serum decreased the number of cells but did not cause any morphologic change or any obvious cell damagc. Bovine capillary endothelial cells exposed to pooled NHS manifested an abnormal morphology, characteristic of nonproliferating endothelial cells. The cells appeared flat with numerous cytoplasmic grains. In contrast, cultures exposed to the inhibitory serum proliferated rapidly and created a confluent monolayer of closely apposed cuboidal cells, representing the normal appearance of these cells when grown under optimal conditions in calf serum in the presence of fibroblasr growth factor. 8

Purification of the ~'mhibitory activity from serum of the patient with perigraft seroma Precipitation of serum proteins with ammonium sulfate at a final saturation of 50% eliminated the inhibitory activity from both the pellet and supematant fractions. However, treating pooled NHS and inhibitory serum with ammonium sulfate at a final saturation of 25%, precipitated 10% and 24% of the total serum proteins, respectively, and the inhibitory activity appeared exclusively in the ammonium sulfate precipitate of the inhibitory serum (Table II).

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Schneiderman et al.

106

Fig. 1. Morphology of HSF and capillary endothelial cells exposed to the inhibitory and normal serum. HSF cells (A, B) and bovine brain capillary endothelial cells (C, D) were grown in the presence of 50% and 25% serum, respectively. A and C were exposed to normal human serum; B and D were exposed to the inhibitory serum. Cultures were photographed on day 6 (Original magnification x 100).

Table I. Effects of serum derived from the patient with perigraft seroma on the proliferation of various cultured cells ( x lO-S cells~35 mm dish) Cell type Autologous fibroblasts Homologous fibroblasts Human smooth muscle Bovine brain capillary endothelium

Concentration of serum (%)

Inhibitory serum*

Normal human serum*

50% 50% 25% 50% 10% 25%

1.35 ± 0.05 2.85 ± 1.25 3.75 3.92 2.82 ± 1.57 2.87 ± 0.67

3.24 ± 0.09* 4.51 _+ 1.72" 5.16" 6.35 0.46 + 0.11"* 0.46 _+ 0.45

Inhibition **/proliferation*** 58 (n = 37 (n = 28 38 613 (n = 623 (n =

2) 7)

3) 3)

*The values presented are mean _+ SD. Net growth in inhibitory serum

**(1

-

-) x 100.

Net growth in normal serum ***Net growth in inhibitory serum x 100. Net growth in normal serum

Ammonium sulfate precipitate (25% saturation) was dissolved and dialyzed against phosphatebuffered saline containing 0.04% CHAPS. To avoid any possible aggregation of the inhibitory factor or its association with other serum proteins CHAPS was added. A previous study and our preliminary experiments show that CHAPS at a final concentration of 0.01% in the growth medium did not affect cell

proliferation. 1° The sample was applied to the sepharose 6B column, and the material was eluted from the column with DPBS containing 0.04% CHAPS. The elution pattern is shown in Fig. 2. Groups of 10 fractions were pooled, and the inhibitory activity of each pool was tested with H S F cultures. Most of the proteins were eluted from the column in fractions 40 to 60, and the peak of

Volume 14 Number 1 luly 1991

Fibroblast inhibitory factor and perigrafl seroma 107

c4 d

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Froction # Fig. 2. Purification of the inhibitory activity by sepharose 6B column. Inhibitory and NHS were precipitated with ammonium sulfate (25%), dissolved, dialyzed and applied to a sepharose 6B column. Fractions were collected, and absorbance at 280 nrn was determined (solid line). The fractions were pooled, concentrated, and their effect on the proliferation of HSF cells at a final concentration of 25% was studied (bars). The pool of tubes 30-39 was concentrated and reapplied to the column. Fractions at 5% final concentration were assessed for inhibitory activity (insert). The column was calibrated by use of molecular mass standards as indicated on the upper part of the figure.

inhibitory activity was demonstrated in the pool of fractions 30 to 39. This active fraction was reapplied to a sepharose 6B column. The eluted fractions, 34 to 43, were pooled in pairs, concentrated (1 ml each), and evaluated for their inhibitory effect. Maximal inhibitory activity was observed in fractions 38 to 39 (Fig. 2, insert). The apparent Mr calculated for the inhibitory factor using molar mass standards was 2 3 0 kDa. Miquots of the', pooled fractions shown in Fig. 2, insert, were applied to SDS-PAGE (Fig. 3). The gel pattern demonstrates a diffuse band with an apparent Mr of 230 kd. N o other contaminating bands could be detected in the gel, indicating the purity of the inhibitory factor obtained at the end of the second run on the sepharose 6B column. The diffuse protein band on the gel is characteristic of glycoproteins. Characterization o f the inhibitory factor Sequential removal of albumin and immunoglobulins by affinity chromatography from the active fractions (tubes 30 to 39), demonstrated that the inhibitory activity was not associated with immunoglobulins or albumin (data not shown). However, the albumin and immunoglobulin-free serum ob-

Table II. Characterization of the inhibitory factor Inhibition of HSF proliferation (%) A m m o n i u m sulfate precipitation 25% saturation 50% saturation Albumin and immunoglobulin-free fraction Concanavalin A b o u n d

29% 0% 60% 28%

Fractions were tested at a concentration o f 25%. T h e percentage o f inhibition was calculated as the relative proliferation in the presence o f the fraction o f the inhibitory serum compared to the proliferation in the presence o f the parallel N H S fraction.

tained by this procedure appeared to have a very potent inhibitory activity on HSF proliferation (Table II). The nature of the inhibitory factor was further evaluated by applying the albumin and immunoglobulin-free fraction to the concanavalin A-sepharose column. The inhibitory factor demonstrated specific interaction with concanavalin A and was eluted from the: column by et-methyl-Dmannoside solution (Table II). This result indicates that the inhibitory factor is probably a glycoprotein.

108 Schneiderman et al.

Fig. 3. Gel electrophoresis of the inhibitory fraction. Lanes 1-4 are samples derived from NHS and lanes 5-8 are samples derived from the inhibitory serum. Lanes 1 and 5, fractions 34-35; Lanes 2 and 6, fractions 36-37; Lanes 3 and 7, fractions 38-39; Lanes 4 and 8, fractions 40-41. Molecular mass standards are marked on the left side of the gel.

DISCUSSION

Inhibition of the proliferation of HSF cells taken from patients with perigraft seroma by autologous serum was demonstrated in vitro by Sladen et al. 6 and Aim et al. 1 Their data strongly support the assumption that a humoral inhibitory factor is responsible for defective proliferation and maturation of fibroblasts, resulting in impaired incorporation of the prosthetic graft into the tissue. In the present study two homologous skin fibroblast cell lines and autologous skin fibroblasts were used to investigate the inhibitory capacity of crude inhibitory serum from a patient with perigraft seroma. The inhibitory serum was tested and compared to a series of individual and pooled N H S that were found to possess the same proliferative properties in cell cultures as "true control serum" (prepared from a patient who underwent the same vascular reconstructive procedure and exhibited norreal incorporation of the prosthetic graft). Proliferation of both fibroblast cell lines was inhibited in

Iournal of VASCULAR SURGERY

the presence of 25% and 50% crude inhibitory. serum. Application of 50% crude inhibitory serum to autologous skin fibroblast cultures and to homologous HSF resulted in proliferation inhibition of 58% and 32%, respectively. These findings suggest that the humoral inhibitory activity is more pronounced in autologous fibroblasts than homologous HSF (by 1.8-foM); however, the inhibitory factor is not specific for autologous fibroblasts, but is rather a generalized fibroblast proliferation inhibitory factor. The effect of crude inhibitory serum on two other cell lines, homologous smooth muscle (human saphenous vein origin) and bovine capillary endothelial cells, was evaluated. Treatment of homologous smooth muscle cultures with 50% crude inhibitory serum resuked in proliferation inhibition of 38%. In contrast to its inhibitory effect, bovine capillary endothelial cell cultures exhibited a striking proliferative effect of 7.1-fold increase in cell number when treated with the inhibitory serum. Bovine brain capillary endothelial cells were shown to actively proliferate when exposed to optimal conditions such as calf serum and fibroblast growth factor. 8 Exposure of these cells to individual and pooled N H S resulted in a limited proliferation, probably because of the inadequacy of N H S and to the lack of fibroblast growth factor. The serum derived from the seroma patient significantly supported the proliferation of the bovine capillary endothelial cells; however, the nature of this stimulation is not dear. Apparently the inhibition of HSF cultures by this serum is not due to a nonspecific cytotoxic factor present in the serum, but rather to a specific factor present only in this serum which has specific target cells. Inhibition offibroblast proliferation is not unique to this described factor but has been shown in other clinical and pathophysiologic conditions. Glucocorticoids have been shown to inhibit fibroblast proliferation in cultures derived from human and mouse tissues. 11'12 H u m a n leukemia cells release a humoral fibroblast inhibitory substance.13 Fibroblast proliferation inhibition has been shown with prostaglandin E2, a product of alveolar macrophages. ~4 Stimulated mononuclear cells release an inhibitory factor to human lung and dermal fibroblasts} s'16 Fibroblast cultures were inhibited when exposed to oxygen tension exceeding 140 mm H g ) 7 Proliferating cultures of 3T3 fibroblast cells treated with medium conditioned by exposure to density-inhibited 3T3 fibroblast cells, show inhibition of growth and division) 8 Experimental data indicate that endothelial cells

Volume 14 Number 1 July 1991

may regulate smooth muscle cell growth. Several substances with antimitotic effect on smooth muscle cells have been identified. For example, heparin-like substances, released from bovine aortic endothelial cells by platelet endoglycosidase, have been shown to inhibit bovine smooth muscle cell proliferation. 19 Exogenous heparin is antimitotic to smooth muscle cells in c u l t u r e , 2° while being a strong enhancer o f the proliferative effect o f basic or acidic fibroblast growth factor on endothelial cells. 21 Constituents o f the extracellular matrix are involved in the modulation of various cell processes including cell proliferation. For instance, heparin sulfate proteoglycans, synthetized by postconfluent smooth muscle cells, have an antiproliferative effect on smooth muscle cells. 22 Heparan sulfate within the extracellular matrix has been shown to store', basic fibroblast growth factor, 23 and the latter may serve as a potential angiogenic stimulus for neovascularization. Our crude inhibitory serum significantly inhibited homologous smooth muscle cell multiplication but had an extremely proliferative effect on bovine capillary endothelial cells. These results should be further investigated in the light o f previous data. At present, we have no grounds to assume that the fibroblast proliferation inhibitory factor is the same agent affecting human saphenous vein s m o o t h muscle or bovine brain capillary endothelial cultures. The present findings suggest that the process o f defective artificial graft incorporation into the tissue is probably more complex than just fibroblast proliferation inhibition. It seems reasonable to assume that other cells are involved in this complex process, namely smooth muscle and endothelial cells. This should be further investigated. Partial purification and characterization o f the molecular properties o f the inhibitory factor were elucidated by routSne techniques including ammonium sulfate precipitation (25% saturation), followed by sepharose 6B gel filtration. Fine fractionation on sepharose 6B column yielded exclusive inhibitory fractions, which appeared as a diffuse band on gel electrophoresis characteristic o f glycoproteins. Both modalities showed an apparent M r of about 230 kd, and it was found to be a glycoprotein by its affinity to concanavalin A. Albumin and immunoglobulin were separated from the inhibitory serum by the use o f a goat anti-(human serum albumin)-sepharose column and goat anti-(human immunoglobulins)-sepharose column, respectively. N o significant inhibitory activity was detected in the immunoglobulins or albumin fractions, whereas the serum devoid o f immunoglobulins and albu-

Fibroblast inhibitory ~kctor and perigrafl seroma

109

min exhibited 60% inkibition o f fibroblast proliferation. These findings contradict the theory o f an immunologic reaction to the implanted graft as the basis for impaired prosthesis incorporation. The authors thank Mrs. Nahid Farzame for her dedicated technical assistance, and Prof. D. Gospodarowicz (University of California, San Francisco, Calif.) and Prof. D. G. S. Thilo-Korner (University of Giessen, Giessen, Federal Republic of Germany) for their kind donation of bovine brain capillary endothelial cells and human saphenous vein smooth muscle cells, respectively. REFERENCES

1. Ahn SS, Machleder HI, Oupta R, Moore WS. Perigraft seroma: clinical, histologic, and serologic correlates. Am ]" Surg 1987;154:173-8. 2. Kaupp HA, MatulewieczTJ, Lattimer OL, KremerJE, Celani VJ. Graft infection or graft reaction? Arch Surg 1979;114: 1418-22. 3. SlizagyiDE. In discussion: Kaupp et al. Graft infection or graft reaction? Arch Surg 1979;114:1422. 4. Blumenberg RiM, GlefandML, Dale WA. Perigraft seroma complicating arterial grafts. Surgery 1985;97:194-203. 5. VollmarJF, GuldnerNW, Mohr W, Paes E. Perigraftreaction after implantation of vascular prostheses. Int Angiol 1987;6: 287-93. 6. SladenJG, Mandl MAI, GrossmanL, Denegri JF. Fibroblast inhibition: a new and treatablecauseofprostheticgraft failure. Perigraftseroma: clinical,histologic, and serologiccorrelates. Am J Surg 1986;149:587-90. 7. GospodarowiczD, BialeckiH, Greenburg G. Purificationof the fibroblastgrowth factor activityfrom bovine brain. I Biol Chem 1978;253:3736-44. 8. GospodarowiczD, Massoglia S, Cheng J, Fujji DK. Effectof fibroblastgrowth factor and lipoproteinson the proliferation of endothelial cells derived from bovine adrenal and brain cortex and bovine corpus luteum capillaries. J Cell Physiol 1986;127:121-36. 9. LaemmliUK. Cleavageof structuralproteins during assembly of the head of bacteriophage T4. Nature 1970;227:680-5. 10. Matuo Y, Nishi N, Mugtmmaa Y, et al. Stabilization of fibroblast growth factor by a non-cytotoxiczwitterionic detergent, (3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonate (CHAPS). In Vitro Cell Dev Biol 1988; 24:477-80. 11. MecoyBJ, DiegelmannRY, KelmanCI. In vitro inhibitionof cell growth, collagensynthesis, and prolylhdroxylaseactivity by triamoinolone acetonide. Proc Soc Exp Biol Med 1980; 183:216-22. 12. KruseNJ, Rowe DW, FujimotoWY, BornsteinP. Inhibitory effects of glucocorticoids on collagen synthesis by mouse sponge granulomas and granuloma fibroblasts in culture. Biochim Biophys Acta 1978;540:101-16. 13. Nagao T, Yamauchi K, Komatsuda M, et al. Inhibition of human bone marrow fibroblastcolonyformation by leukemic cells. Blood 1983;82:1261-5. 14. Rennard SI, Bitterman PB, Crystal RG. Current conceptsof the pathogeuesis of fibrosis: lesions from pulmonaryfibrosis. Prog Clin Biol Res 1984;164:359-77. 15. Elias JA, Rossman MD, Daniele RP. Inhibition of human

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16.

17. 18. 19. 20.

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lung fibroblast growth by mononuctear cells. Am Rev Respir Dis 1982;125:701-5. Korn JH, Halushka PV, LeRoy EC. Mononuclear cell modulation of connective tissue function: suppression of fibroblast growth by stimulation of endogeneous prostaglandin production. J Clln Invest 1980;65:543-54. Balin AK, Fisher AJ, Carter DM. Oxygen modulates growth of human at physiologic partial pressures. J Exp Med 1984; 180:152-66. SteckPA, Voss PG, Wang JL. Growth controlin cultured 3T3 fibroblasts. J Cell Biol 1979;83:562-75. Castellot JJ, Favrau LV, Karnovsky MI, Rosenberg RD. Inhibition of vascular smooth muscle cell growth by endothelial cell-derivedheparin. J Bioi Chem 1982;257:11256-60. Hoover RL, Rosenberg R, Haering W, Karnovsky ML

Inhibition of rat smooth musde celt proliferation by heparin. In vitro studies. Circ Res 1980;47:578-83. 21. Gospodarowicz D, Ferrara N, Schweigerer L, Neufeld G. Structural characterization and biological fimctions of fibroblast growth factor. Endocrine Rev 1987;8:95-114. 22. Fritze LMS, Reilly CF, Rosenberg RD. An anfiproliferative heparan sulfate species produced by postconfluent smooth muscle cells. J Cell Biol 1985;100:1041-9. 23. Folkman J, Klagsburn M, Sasse 1, Wadziniski M, Ingber D, Vlodavsky I. A heparin-binding angiogenic protein basic fibroblast growth factor is stored within basement membrane. Am J Pathol 1988;130:393-400. Submitted July 2, 1990; accepted Jan. 16, 1991.