Identification of new collagen formation with 125I-labeled antibody in bovine pericardial tissue valves implanted in calves

Nucl. Med. Lib/. Vol. 13, No. 4, pp. 413422, hf. .I. Radiaf. Appl. In.~um. Part B Printed in Great Britain. All rights reserved

1986 Copyright

0

0883-2897/86 $3.00 + 0.00 1986 Pergamon Journals Ltd

Identification of New Collagen Formation with 12’1-Labeled Antibody in Bovine Pericardial Tissue Valves Implanted in Calves M. K. DEWANJEE, S. K. SINGH, P. H. WOOLEY, S. T. MACKEY, E. SOLIS and M. P. KAYE Sections of Diagnostic Nuclear Medicine, Immunology, and the Department of Surgery, Mayo Clinic, and Mayo Foundation, Rochester, MN 55905, U.S.A. Failure of bovine pericardial tissue valve used in young patients may be due to a slow rejection process. Polyclonal anticollagen (Type I) antibody (IgG) was made in rabbits and purified by protein A atIinity column. Two milligrams of IRG was labeled with 2 mCi of “‘1 bv the Iodoaen method. Free iodide was separated by G-10 Column. A%inityof “‘1-1gGwas checked by radioimmuno&say. Two hundred and fifty microcuries of “‘1-1gG was injected in calves immediately after tissue valve implantation, and the calves were killed 4 h post-injection. After harvesting the valve, each of the three leaflets was separated into four zones, and radioactivity in each section was mapped with a y counter. The radioactivity in tissue valve section was compared to that of normal aortic valve. The sections of tissue valve retain five to ten times more ‘251-IgGthan control aortic valve. Iodine-IgG thus provides a sensitive technique for determination of residual antigenicity in tissue valve.

Introduction The major limitation of mechanical prosthetic valves is their propensity for thrombus formation and embolization.(‘q2) Postmortem light and electron microscopic studies have shown thrombotic material on the sewing ring of prostheses and on damaged perivalvular cardiac tissie.od) Patients having mechanical cardiac valvular prostheses need continual anticoagulation therapy to reduce thromboembolic complications. Valves made of biologic materials e.g. fascia lata, dura mater, porcine aortic valves, or bovine pericardium were investigated during the late 1960s and early 1970s. Bioprosthetic valves made of glutaraldehyde-fixed porcine aortic valve and bovine pericardium were considered to be acceptable alternatives to mechanical valve prostheses. Unfortunately, as demonstrated by extensive clinical and pathological studies, these tissue valves undergo degeneration and calcification (intrinsic calcification in collagen and extrinsic calcification in adherent thrombus), especially in children and adolescents.(i2-I’) Supported in part by Research Grant HL-28974 from the National Institutes of Health. Address correspondence to: Mrinal K. Dewanjee, Ph.D., Mayo Clinic, 200 First Street S.W., Rochester, MN 55905, U.S.A. 413

The reasons for degeneration and calcification of tissue valve prostheses have been studied in valve explants from young sheep, calves and children. In the past, partial fibroblast coverage of tissue valve and vascular graft and collagen (Type I) formation was studied with histochemical techniques.(49) Considering the higher sensitivity of tracer technique, in this study, we used r2%labeled collagen antibody as a probe to study new collagen formation over old fixed collagen(‘“2*) in the tissue valve in a calf model. The new collagen synthesized by the proliferating fibroblast on old collagen may be more thrombogenic than the crosslinked collagen and may be responsible for the ongoing platelet thrombosis and calcification of tissue valve prosthesis especially in young animals.

Materials and Methods A. Preparation and purification of rabbit immunoglobulin against Type I collagen

Three milligrams of collagen in 1.5 mL of 0.1 M acetic acid (bovine skin Type I collagen, Sigma Chemical Co.) was emulsified with 1.5 mL of complete Freund’s adjuvant. One and a half milliliter was injected subcutaneously at two sites of New Zealand albino rabbits. This procedure was repeated twice in the first week. After 3 weeks rabbits were bled (twice a week) by arterial (ear) puncture. An amount of

414

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DEWANJEE et al.

RADlOlODlNATlON

lodogen

(methylen chloride. 7 mgimlj

b:~i

i

OF PROTEIN (lodogen)

‘*51-iodide, 2 mCi

7 b:I

2-3 mm

Borate buffer, 7.8 pH @ mix vortex PECT Protein (2-3 min) (loo-500 pg) (1 ml) 0.1-0.2 ml

Fig. 1. Radioiodination of rabbit antibody against bovine skin Type I collagen with Iodogen technique. The transfer of oxidized iodinated species to protein in a separate tube prevents direct oxidation of antibody with Iodogen.

2&25 mL of blood was collected in a sterile tube, allowed to clot overnight, and serum was separated by centrifugation and stored at -70°C. The immunoglobulin was precipitated with an equal volume of 50% ammonium sulfate (cold) in a cold room. After 2-3 min of stirring the precipitate was separated by centrifugation (2OOOg, 10 min, 4°C). The precipitate was ,dissolved in minimum volume of water and dialyzed overnight at 4°C. The immunoglobulin was then affinity purified on a protein A column (Pharmacia Fine Chemicals). B. Radioiodination of immunoglobulin(29) This procedure is shown schematically in Fig. 1. [‘251]sodium iodide (1.5-2.0 mCi, Amersham Co., Arlington Heights, Illinois) diluted with 0.1 mL of 0.05 M phosphate buffer (pH 7.4) was added to Iodogen coated polypropylene vial (1 mL) containing 20 pg of Iodogen prepared as follows. Iodogen (5 mg) was dissolved in 5 mL of methylene chloride solution; aliquots of 20 PL were transferred to the methylene chloride washed polypropylene vials. They were vortexed for 30 s for coating the inside of the vial and left in the flow hood overnight. The vials were capped and left in the refrigerator at -30°C. The vial was vortexed for 3 min after addition of radioiodide. The oxidized iodinated species were transferred to the immunoglobulin (l-2 mg) in 0.5 mL of 0.05 M phosphate buffer in a separate tube and vortexed. After 5 min of incubation, the mixture was transferred to the buffer-washed disposable sterile Sephadex column (PD-10 column containing 9 mL of G-25 M Sephadex gel stabilized with 0.05% merthiolate solution, Pharmacia Fine Chemicals, Piscataway, New Jersey). The column was eluted with 0.05 M phosphate buffer. The radioactivity appearing in 2-4mL fractions was pooled. Measurement of radioactivity in the column showed an adsorption of 5-10%. The labeling efficiency of lZ51-IgG was consistently

60-75%. The pooled ‘251-IgG was mixed with 50 mg of bovine serum albumin and diluted to a concentration of 400 pCi/mL of phosphate buffered saline. The aliquots were immediately frozen at -70°C. Aliquots were used within a pe@od of 10 days post-labeling. At 60 days some cloudy sediment was observed after thawing, and a large amount of denatured ‘251-IgG was adsorbed by the test tube. The novelty of this transfer technique is that the proteins never face the oxidizing agent (Iodogen) and the chance of oxidative damage to sulfur containing sensitive amino acid residues in the protein is minimal. C. Radioimmunoassay of antiserum with ‘**I-Staph A and evaluation of ‘251-immunoglobulin by autoradiography The affinity of ‘251-labeled immunoglobulin was checked by RIA. Twenty micrograms of collagen (150 pg/mL in 0.1 M acetic acid) was transferred to PBS washed polystyrene tubes; collagen was then washed three times with PBS, then incubated for 4 h (4°C) with 0.2% bovine serum albumin in PBS and washed twice with PBS. Five hundred microliters of 0.2% BSA in PBS along with 1OpL of test serum were added to collagen. ‘251-Staph A (20 p L) (NEN Co.) was incubated for 45 min at 4”C, washed three times with PBS, and the radioactivity was measured with a y counter. Antibody dilution assay was performed with a fixed amount of collagen and aliquots of diluted ‘251-antibody. Immunoreactivity of “‘I-antibody was high after radioiodination. The presence of impurities in the ‘251-labeled IgG was checked by SDS (0.05%) polyacrylamide (lo%, Biorad Co.) slab gel electrophoresis.@) The 1251-IgG samples were treated with 5% mercaptoethanol (dissociation of light and heavy chain by reduction) by heating for 5 min at 100°C and sediment removed by

Autoradiography of 1251-lgG (Type 1 collagen)

-Origin

-Heavy chain

-Light chain

Fig. 2. Autoradiograph of electrophoretogram of reduced ‘251-labeledcollagen-antibody. Two prominent bands (heavy and light chains of IgG) are evident.

415

Fig. 5. (a) Scanning electron bundles. (b) Rough inflow

Fig. 6. Scanning

electron

micrograph of smooth outflow surface with thinner highly knitted collagen surface with thicker collagen bundles. This rough inflow surface is less susceptible to fibroblast coverage.

micrograph

of tissue valve explant. Note collagen from host fibroblast. 416

coverage

over inflow surface

417

Identification of new collagen formation

opened, the mitral valve excised and the valve prosthesis was placed (Fig. 3). Mean cardiopulmonary bypass time was 35-40 min. When the calf was awake and breathing on its own the monitoring lines and chest tube were removed. Each animal received perioperative systemic antibiotics (1.2 million units of benzathine penicillin G: Bicillin). Heparin was not reversed with protamine. E. Biodistribution of “‘I-antibody in calves

Fig. 3. Implantation of 25 mm pericardial tissue valve in mitral annulus in a calf heart.

centrifugation at 11,000 g for 5 min. Aliquots (5 FL) were applied to the slab gel and electrophoresed for 4 h at 110 V with the Laemmli discontinuous system. The slab was dried for 2 h, placed in contact with x-ray film (Kodak X-O mat) and exposed for 2 h. The autoradiography of electrophoretogram (Fig. 2) indicates the presence of radioactivity at the site of application (origin) and two prominent bands (heavy and light chains). There is the presence of a diffuse band near the heavy chain. This impurity might affect the results of biodistribution to a small extent, but the relative radioactivity of prosthesis (leaflet/aortic valve ratio) may not be affected. ‘*‘I-labeled antibody was administered intravenously into healthy rabbits (New Zealand Albino: 4-5 kg), five mongrel dogs (22-28 kg), and five Holstein calves (40-50 kg). Serial blood samples (3-5 mL) were collected in preweighed heparinized tubes. The radioactivity in all tubes was determined with a y well counter. From the blood volume, injected ‘*‘I-antibody (75-200 PCi) and radioactivity in the blood samples, the percentage of injected radioactivity in blood at different times after administration were calculated with a computer (IBM-PC). In the calculation of blood volume, it was assumed that the average blood volume in rabbits, dogs, and calves amounted to 7, 9 and 7% of the body weight, respectively. D. Surgical implantation of tissue valves

Twenty calves (40-50 kg) underwent mitral valve replacement with 25 mm Ionescu-Shiley tissue valve prostheses. The calves were anesthetized with ether, maintained on halothane, supplemented with succinylcholine and anticoagulated with heparin (300 units/kg). Two pericardial patches (5 x 5 cm) were placed subcutaneously under the abdomen at the time of valve surgery. A left thoractomy was performed and cardiopulmonary bypass established between the right atrium and femoral artery. Using moderate systemic hypothermia and infusion of cold crystalloid cardioplegic solution the left atrium was

The calves were heparinized and killed with an overdose of sodium pentobarbital. Samples of tissue from the heart, lungs, liver, spleen, kidneys, blood, brain, skeletal muscle, bone and marrow were obtained and weighed. It was assumed that 40, 10,7 and 2% of body weight correspond to skeletal muscle, bone, skin, and marrow respectively. From these data the distribution of radioactivity in the organs and whole body was determined with a triple-channel automatic y well counter (Beckman Gamma 8000). The window of the spectrometer was adjusted to include the 28-, 35-, and 63-keV peaks of ‘*‘I. The adherent thrombus on the valve was removed and the prosthesis was separated into the components: leaflets, posts and sewing ring. For measuring the regional distribution the leafiets were cut into four zones; all these samples were weighed on a microbalance. Subcutaneous patches were removed and treated in a similar fashion. From the radioactivity in these zones of leaflets the ratio of radioactivity in these zones and other samples of heart valves, with respect to that of control aortic valve, was calculated. F, Scanning electron micrograph of pericardial surface

Pericardium obtained from a slaughterhouse was fixed in Trump’s buffer and coated with goldpalladium alloy. The rough and smooth surfaces of pericardium and tissue valve explant were scanned with scanning electron microscope (ETEC Autoscan). Representative sections were photographed.

Results The blood clearance of ‘251-labeled immunoglobulin was checked in three species of animals (Fig. 4). Fifty to sixty percent of administered ‘2SI-IgG cleared from blood soon after injection. The rest cleared slowly with multiexponential components; the fastest clearance was observed in dogs (heterologous antiserum clearance half-life of longest components was approximately 50 h). The slowest clearance was observed in calves (homologous antiserum clearance half-life of longest component was approximately 180 h), and the clearance half-life in rabbits was in between these two extreme values (approx. 90 h). The biodistribution of ‘*‘I-antibody in calves (Table 1) indicates that most of the radioactivity (approx. 20%) at 24 h post-injection and surgery is in

M. K. DEWANJEE et al.

418

I

1 0

30

60

120

90

150

Time post-injection (hour)

Fig. 4. Blood clearance (percent of injected dose) of ‘251-labeled antibody in normal calves, dogs, and rabbits. Clearance is faster in dogs than calves or rabbits.

the blood, about 6% is in the skeletal muscle, and about 5% is in the lungs and skin, respectively. The connective tissues of skin, bone, skeletal muscle and lungs are the major sources of collagen in the body. At 14, 30 and 90 days post-valve implantation less radioactivity (lO--14%) was observed in the blood, a slightly higher amount in skeletal muscle and slightly less in lungs. At 24 h post-surgery, the increased lung uptake noted might represent increased collagen turnover rate and edema. This lung uptake was found higher than liver at all time frames. In the tissue valve the major fraction of ‘Z51-antibody was found in the sewing ring, leaflets and perivalvular tissue at 1, 14,30 and 90 days. In the leaflets and perivalvular tissue a smaller amount is incorporated, although more fibroblast were observed in the healing phase (l-3 weeks). This reduced uptake in the perivalvular tissue and sewing ring might be due to decreased vascularity. The relative binding and retention of ‘251-antibody to different zones of leaflets (Fig. 7), sewing ring, thrombus and perivalvular tissue of components of

tissue valve and that of subcutaneous pericardial patch implants is shown in Table 2. In the initial phase (1 day study) there is a possibility of crosslinking of ‘*‘I-antibody to crosslinked collagen in pericardial leaflet due to the presence of traces of residual glutaraldehyde. In the free edge zone there is a continuous increase in binding reflecting increase in collagen coverage by the growing fibroblast. In the flexion zone there is a continuous decrease. Adherent thrombus in the flexion zone incorporates more “‘I-antibody than native aortic valve. The continuous collagen growth from the attachment zone may be responsible for this enhanced binding. The attachment zone at 14 and 30 days binds more “‘I-antibody, reflecting new collagen synthesis. The increase in binding at 14 and 30 days at the outer and inner sewing ring also reflects the increase in and binding with new fibroblast growth collagen-antigen. Subcutaneous implants also bind “‘I-antibody, although decreased vascularity may be responsible for reduced uptake. The scanning electron Jmicrograph of smooth (outflow surface) and rough (inflow) surface of pericardium is shown in Figs Sa and b, respectively. Four to five times more platelets were trapped in the rough surface. Scanning electron micrograph of tissue valve explant showed new collagen from host fibroblast on fixed bovine pericardial leaflet. The new collagen fiber appears thicker than the old one (Fig. 6).

Discussion Norma1 pericardium is composed of Type I collagen (80%) and the rest is elastin. The proteoglycans and mesenchymal cells which are originally present in the pericardium are lost during processing of pericardium. In the viable pericardium these viable cells control the influx and efflux of calcium ion. In the glutaraldehyde-fixed pericardium no such calcium regulator is present.

Table I. (Mean f SD of percentage of injected dose) of ‘2’1-labeled rabbit antibody against collagen in tissue valve implanted calves at 24 h post-injection and I, 14, 30 and 90 days post-implantation I day (n = 5) Liver Spleen Lungs Kidneys C0rteY. Medulla Heart Skeletal muscle Brain Blood Skin x (IO_” Tissue valve

14 days (n = 5) 30 days (n = 5) 90 days (n = 5)

2.5 f 0.6 0.9 f 0.3 4.8 * I.7 0.7 * 0.3 0.6 f 0.3 0.2+0.1 0.5 f 0.2 6.1 f 2.4 0.06 f 0.02 20.3 + 5.5 4.6 f 3.5

I.5 f0.7 0.3 + 0.1 3.5 5 2.6 0.9 f 0.2 0.8 f 0.1 0.2 f 0.1 0.4ztO.l 9.1 f4.5 0.07 * 0.04 10.0 k 6.2 6.5 + 3.2

2.3 t 0.8 0.5 f 0.2 3.9 f I.3 1.0+0.1 0.8 fO.l 0.2 f 0.1 0.4 * 0. I 8.4 k 3.6 0.06 + 0.03 13.9 f 5.8 6.2 f I.1

2.2 + 0.7 0.4 f 0.3 3.7 * 2.0 1.3 * 0.5 I.1 +0.5 0.2+0.1 0.4ItO.l 7.9 + I .7 0.05 + 0.02 10.9 f 4.7 8.6 k 1.8

18.1 k6.6

18.5 + 8.1

9.5 + 5.1

10.9 f 4.7

Components

Leaflets Sewing ring Perivalvular tissue

of tissue

valve ( x

IO-‘)

8.3 + 3.0 9.5 f 3.9

10.6 k 5.2 7.4 f I.8

4.3 f 2.3 4.8 + I.6

6.9k4.1 3.6 f I. I

2.9 _+1.8

2.2 f I.7

1.0 +0.4

0.5 f 0.2

Identification of new collagen formation Table

419

f SD) of relative radioactivity (‘lJI-antibody) of regions of leaflets and components of valve to that of aortic valve in tissue (bovine pericardium) valve implanted calves at 1, 14, 30, and 90 days post-implantation

2. (Mean

Radioactivity of region of leaflet/radioactivity of aortic valve Components of tissue valve

1day

Free edge zone Central zone Flexion zone Thrombus Attachment zone Outer sewing ring Inner sewing ring Sewing post Perivalvular tissue

14 days

30 days

90 days

5.73 + 3.75 5.87 + 2.43 4.94 + 2.54 2.125 1.72 I .57 f 0.27 1.16kO.20 0.74 + 0.19

7.98 * 3.31 f 4.20 f 3.50 f 6.90 i 4.22 f I .04 * 1.22 f

4.9 I 2.13 3.24 2.10 3.03 2.80 0.39 1.16

8.41 + 6.10 + 3.76 f 5.19 f 6.68 f 1.45 f 4.60 + 4.01 f

6.49 4.27 3.13 3.29 4.87 0.83 2.95 2.35

6.67 k4.12 3.91 * 2.15 3.37 f 1.98 12.42 + 1.29 3.43 + 2.51 0.96 + 0.22 1.62f 0.60 0.84 f 0.63

1.55 + 0.20

0.94 f 0.28

I .27 f

0.67

0.98 f 0.29

Subcutaneous pericardial implanrs Fixed pericardium Diphosphonate-bonded wricardium

3.48 f 1.09

1.17 f0.57

2.84 + 2.32

1.35f0.81

I .60

1.19 f0.39

1.89f I .39

1.59 + 1.04

3.84 f

When blood comes in contact with the collagen fiber (crosslinked) the platelet micro-thrombus forms in the junction of leaflet and sewing ring; part of this thrombus embolizes, a fraction forms vesicles and calcifies (Fig. 8). In addition, due to the antigenic determinants of collagen in certain individuals there is a possibility of activation of granulocyte and macrophage. The collagenase released might be another source of direct degeneration of collagen fiber. Directly exposed collagen may also cause autoimmune type reaction, probably cell mediated as observed in arthritis, progressive systemic sclerosis and glomerulonephritis. Further studies are necessary to understand the role of cell-mediated activities in tissue valve degeneration.

There are currently 13 types of collagen identified by the biochemists, o’~) although Type I collagen is the predominant structural protein of the extracellular matrix in the connective tissue (bone, skin, blood vessel, pericardium, muscle). The turnover of plasma proteins can be easily studied by the labeled proteins using multiple blood sampling. Due to the difficulty of solubilizing and sampling, no such studies can be performed using labeled structural proteins (collagen, elastin, laminin), although these studies have been carried out using fluorescein and ‘251-labeled antibodies to collagen and laminin and measurement of specific activity using 3H- or 14C-labeled proline, 14C-glycine and

hydroxyproline.

In patients

increase

of hydroxy-

COLLAGEN COVERAGE OF LEAFLETS WITH FIBROBLAST Crosslinking (OCXL) and Binding of lz5 I-Ab With Old (OCB) and New Collagen (NCB) Fibroblast

growth aortic valve)

I-Ab-Ag Crosslinking, (GA-OCXL) Old Collagen Binding (OCB)

I-Ab-Ag Binding (OCB, NCB)

I-Ab-Ag = “‘I-Ab bound collagen. -coverage by new collagen I-Ab = “‘l-antibody (rabbit-type 1 collagen)

I-Ab-Ag Binding (OCB, NCB) Ag = Antigen (collagen) GA = Glutaraldehyde FE = Free Edge

I-Ab-Ag Binding (OCB, NCB) CZ = Central zone FZ = Flexlo” zone AZ = Allachmenl zone

Fig. 7. Evaluation of collagen coverage of pericardial leaflets with ‘2SI-antibody. Increase of relative radioactivity (leaflet zone/aortic valve) ratio as shown by a number in each zone indicates higher ‘*rI-binding in the free edge and attachment zone due to new collagen coverage by proliferating fibroblast along sewing ring and posts. The arrows indicate the site of new collagen-fiber-profile synthesized by proliferative host fibroblast over old glutaraldehyde crosslinked collagen. ‘25I antibody indicates the presence of Type I collagen. This new bare collagen surface may be susceptible to platelet thrombosis and calcification.

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THROMBOSIS AND CALCIFICATION (Tissue Valves) RBC

Platelet

a. Drugs (Th. Cal. Inl) b. Glutaraldehyde (toxicity) (XL-plasma proteins)

L

1

GC = Granulocyte NSI = Nucleation Site Inhibitor NSP = Nucleation Site Promotor PA = Plasrntnogen Activator RBC = Red Blood Cell SW = Smooth Muscle Cells Th = Thrombosis XL = Crosslinked

proteOglyCanS

(NW

1

Cai = Calcihcation CD = Cell Death EC = Endothelial Cells FBR = Foreign Body Response FGF = Fibroblast Growth Factor Inf = Inlection M = Macrophage

Ag-Ab-Cl = Antrgen (collagen Type I)-Antibody-Complement

1

Interaction of glutaraldehyde-lixed perrcardium wrlh plasma proterns, electrolytes, cellular elements of blood and connective tissue

Fig. 8. Mechanism of thrombosis and calcification of tissue valve prosthesis leading to stenosis and degeneration of collagen fiber. This problem of calcification is severe in young patients and less intense in adult patients.

proline

in urine represents

higher collagen

turnover.

Since the renal, intestinal, splenic and hepatic tissue contain fenestrated endothelium, these labeled antibodies can reach these tissues even if the antibody molecule (IgG, approx. 150,000 dalton) is large. In addition, several diseases of connective tissues and the associated inflammation, increase the turnover of collagen locally. Radioiodination of antibody using Iodogen transfer technique is a simple, fast and reproducible method for protein labeling. The vials were found suitable for iodination of fibrinogen, lipoproteins, serum albumin and immunoglobulins 6 months postcoating. Since protein does not come in contact with Iodogen, the chance of oxidative damage to disulfide bonds is low. In addition, 8&85% immunoreactivity of labeled antibody was retained in collagen binding assay. Few data are available on the affinity of antibodies produced against collagen or procollagens. All polyclonal antisera react with several antigenic determinants in the terminal and helical regions of collagen chains; the affinity constant for antibodies of amino terminal region (10” Ljmol) is several orders of magnitude higher than that of the helical region. The amino acid sequence of N-terminal of alphal(1) chain of calf collagen: Glu-Leu-Ser-Tyr-Gly-Tyr-

Asp-Glu-Lys-Ser-Thr-Gly-Ile is found to react with rabbit antiserum. Several investigators(32,33) have made antibodies against collagens. Rabbits produce antibodies that react with terminal regions of the molecule; antibodies from guinea pigs, rats, mice, sheep and chicken react with antigenic determinants within the triple helix. Compared to native triplehelical collagen, the denatured collagens are weaker antigens. Soluble collagen is more immunogenic than crosslinked collagen. Upon crosslinking of soluble plasma proteins with residual glutaraldehyde, the tissue valve may become more immunogenic. Production of antibodies to collagen in rabbits requires the presence of complete Freund’s adjuvant at least for the first injection. The distribution of the various coliagens and procollagens in different organs and tissue samples was found to change considerably during embryonic development and pathological changes of fibrosis (lung, skin, liver and blood vessels).04*3s) Previous studies indicate that antibodies to collagen are cytotoxic for fibroblast.(‘i) Antisera to collagen specifically enhance the uptake of collagen by macrophage. (42)Serological studies indicate the presence of antibodies to denatured collagen in rheumatoid arthritis(43) and chronic liver disease. Implantation of bovine pericardial tissue valves in

421

Identification of new collagen formation humans may expose new collagen antigen to the immune system. Autoimmune reactions against collagen due to B cells or regulatory T cells and/or effector T cells have been observed in patients with progressive systemic sclerosis and rheumatoid arthritis. T cell reactivity is detected by skin test, by binding of labeled antigens to lymphocytes and by collagen-activated T-cell factor inhibiting macrophage migration. Generation of rheumatoid arthritis in mice and rats has been induced by injection of Type II collagen. ‘40++ Transfer of antibodies and lymphocytes (B and T cells) from sick into healthy animals also produces arthritic symptoms. Our data suggest that even glutaraldehyde-fixed pericardial collagen may provoke immunological response. Due to direct collagen formation by regenerating fibroblast, total immunoreactivity of the collagen in the leaflet can be quantified with the “‘I-antibody. An index of collagen coverage is obtained from the radioactivity ratio of different zones of leaflet and thrombus with respect to that of control aortic valve. The inhomogeneous binding of “‘I-antibody shows the differential rate of regional collagen coverage in tissue valve in calves, and its progression with time. We have recently quantified the rate of progression of platelet thrombus formation in pericardial tissue valves implanted in calves with “‘ln-labeled platelets.@ 9’ In addition, we also quantified the rate of calcification of collagen and platelet thrombus. The rate of calcification of thrombus and collagen is very similar. We also observed the progression of thrombosis and calcification and collagen fiber degeneration. The former process can be explained by the synthesis of new Type I collagen from the proliferative fibroblast progressing from the base of the valve to the free edge of the leaflet. The latter process might be due to the degeneration of collagen fiber by the infiltrating granulocytes. The foreign collagen antigen might generate collagen antibodies in the host. These antigen-antibody complexes might activate complement-l (classical pathway). This complex might be chemoattractant for granulocytes, monocytes, and macrophages. The extent of this leukocyte infiltration in the patient depends on the specific difference between the host (i.e. human) and tissue implant (bovine pericardial or porcine aortic valve, collagen Type I). ‘251-labeled antibodies provide a powerful technique for the evaluation of this process of collagen (Type I) coverage of tissue valves. Nonspecific binding in the early phase might be due to binding of ‘251-collagen antibodies to pericardial collagen. In addition, residual glutaraldehyde might also crosslink ‘251-antibody to native crosslinked collagen. We have compared the binding of ‘251-labeled albumin, fibrinogen and antibody to fresh and glutaraldehyde-fixed saline-washed pericardium. In spite of repeated washing, the fixed pericardium retained two to threefold higher level of all of the

labeled proteins. Since “‘I-antibody was injected immediately post-valve implantation, we think residual glutaraldehyde will increase binding by crosslinking in addition to the antigen-antibody reaction; although due to complexity of plasma protein environment, continuous washout of glutaraldehyde and ongoing thrombosis, their exact contribution is difficult to ascertain. Early binding and crosslinking may thus account for a twofold to sixfold increase in binding over native aortic valves. ‘251-antibodies bound to new collagen at 30 and 90 days in the free edge of leaflets increased threefold to eightfold over that of native aortic valves (Fig. 7). This significant increase over nonspecific binding definitely suggests the presence of new Type I collagen synthesized by proliferative fibroblasts. Fibroblasts also synthesize Type III collagen; we did not check the crossreactivity of ‘251-polyclonal antibodies with Type III collagen. Our studies of ‘251-binding to thrombus show a fourfold to twelvefold higher binding to thrombus over that of native aortic valves (Table 2). This labeled antibody thus provides a simple technique for mapping collagen coverage of tissue valve prostheses. This explains the ongoing pathologic processes of thrombosis and calcification. Acknowledgements-The

authors highly appreciate the technical assistance of Mr A. Hildestad and Mr S. Chowdhury and excellent typing assistance of Mrs Judy Ashenmacher, MS Mary Mohlke, and Mrs Randi Fravert. The Ionescu-Shiley valves were kindly provided by Dr Jay Lenker and MS Caren Langhammer Irvine, California.

from Shiley Inc.,

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