Measurement of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Blood and...

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Measurement of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Blood and Tissues

Clinical and Experimental Applications

STANLEY ZUCKER

,

a,b

MICHELLE HYMOWITZ,

a

CATHLEEN CONNER,

a

HOSEIN M. ZARRABI,

a

ADAM N. HUREWITZ,

b

LYNN MATRISIAN,

c

DOUGLAS BOYD,

d

GARTH NICOLSON,

e

AND STEVE MONTANA

a

a

Departments of Medicine and Research, Veterans Administration Medical Center, Northport, New York 11768, USA

b

State University of New York, Stony Brook, New York 11794, USA

c

Vanderbilt University, Nashville, Tennessee 37232, USA

d

University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA

e

Institute for Molecular Medicine, Huntington Beach, California 92649, USA

ABSTRACT: The balance between production and activation of MMPs andtheir inhibition by TIMPs is a crucial aspect of cancer invasion and metastasis.On the basis of the concept that MMPs synthesized in tissues seep into thebloodstream, we have examined MMP levels in the plasma of patients withcancer. In colorectal, breast, prostate, and bladder cancer, most patients withaggressive disease have increased plasma levels of gelatinase B. In patientswith advanced colorectal cancer, high levels of either gelatinase B or TIMPcomplex were associated with shortened survival. We propose that these assaysmay be clinically useful in characterizing metastatic potential in selected kindsof cancer. In rheumatoid arthritis and systemic lupus erythematosus (SLE), se-rum and plasma levels of stromelysin-1 were ~ 3–5-fold increased. Fluctuatingserum stromelysin-1 levels in SLE did not correspond with change in diseaseactivity. In SLE, stromelysin-1 may be a component of the chronic tissue repairprocess rather than being responsible for inciting tissue damage. On the basisof these observations, we conclude that measurement of plasma/serum MMPand TIMP levels may provide important data for selecting and following pa-tients considered for treatment with drugs that interfere with MMP activity.

ROLE OF MMP

S

, TIMP

S

, AND EMMPRIN IN CANCER INVASION AND METASTASIS

Production and activation of matrix metalloproteinases (MMPs) in tumors is animportant aspect of cancer invasion and metastasis. Tissue inhibitors of metallopro-

b

Address for correspondence: Stanley Zucker, M.D. (Mail Code 151), VA Medical Center,Northport, New York 11768, USA. Phone, 516/261-4400, ext. 2861; fax, 516/544-5317; email:[email protected]

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S

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S

teinases (TIMPs) counteract the proteolytic activity of MMPs. A delicate balance be-tween activation and inhibition of MMPs (gelatinases, collagenases, stromelysins,matrilysin) at the invasive edge of a cancer controls the degradation of extracellularmatrix (ECM) and subsequent dissemination of the cancer. A correlation betweenhigh levels of MMPs in cancer tissues and aggressive behavior of experimental tu-mors has been repeatedly demonstrated. Paradoxically, TIMP levels are often in-creased in cancer tissues, but this compensatory increase presumably is inadequateto counteract ECM degradation.

1,2

MMPs are also involved in many aspects of nor-mal tissue development and repair involving cell motility, release of growth factorsbound in tissues, and remodeling of the ECM.

A basic limitation in many studies performed to date is the uncertainty of whetherMMPs are involved in the initiation of tissue damage in a disease process or are in-volved in the repair mechanism. Likewise, it is unclear whether TIMPs are producedin response to increased MMP production or whether they are independently con-trolled. In this regard, we are only beginning to understand the relationships betweenMMP and TIMP production by epithelial, stromal, and endothelial cells in disease-targeted tissue. At present, it is appreciated that the concept of MMPs versus TIMPsas the controlling factors in cancer invasion and metastasis is an oversimplificationof a much more complicated series of events.

Although experimental studies initially suggested that cancer cells themselvesproduce the matrix metalloproteinases required for degradation of the extracellularmatrix, more recent studies using

in situ

hybridization techniques have indicated thatmost MMPs are produced by stromal cells within human tumors (breast, gastrointes-tinal, lung, prostate), rather than by the cancer cells themselves. Gelatinase A(GLA),

3–7

collagenase-1,

8

stromelysin-3,

9–11

collagenase-3, and membrane type1-MMP (MT1-MMP)

9,11

have been identified in stromal fibroblasts in distinct pat-terns, especially in proximity to invading cancer cells.

12

Gelatinase B (GLB) hasbeen localized to inflammatory cells (macrophages and neutrophils).

4,5,13,14

Stromelysin-1 and stromelysin-2 were infrequently identified in human tumors.Matrilysin represents the major exception since this MMP is produced by carcinomacells, rather than by stromal cells.

15,16

Membrane type 1-MMP (MT1-MMP) is recognized to be an important physio-logic activator of progelatinase A in benign and malignant conditions.

17–19

Thephysiologic activation mechanism for other secreted MMPs remains to be deter-mined. By means of an immunohistochemical technique, MT1-MMP protein hasbeen reported to be primarily localized in and on gastric carcinoma cells along withgelatinase A.

20

The mechanism of transport of MT1-MMP to the cancer cell surfaceafter synthesis in stromal cells remains to be elucidated. The functional activity ofMT1-MMP after release from the cell surface is also uncertain.

TIMP-1, TIMP-2, TIMP-3, and TIMP-4 form physiologically irreversible com-plexes with all types of activated MMPs in the amino terminal portion of the en-zymes. In addition, TIMP-1 forms a specific complex with latent gelatinase B, andTIMP-2 forms a specific complex with latent gelatinase A in the carboxy terminalregions of the respective enzymes. These unique latent complexes lead to stabiliza-tion of the enzyme activation mechanism. Many types of cancer cells and peritumor-al mesenchymal cells secrete increased concentrations of both TIMPs andgelatinases leading to the formation of complexes of MMPs and TIMPs extracellu-

214 ANNALS NEW YORK ACADEMY OF SCIENCES

larly.

3,21

These complexes subsequently leach into the blood stream, where they canbe identified by immunoassays.

22

An explanation for the role of stromal cells in cancer has come from the seminalwork of Biswas,

et al.,

who demonstrated that carcinoma cells produce a stimulatoryfactor,

E

xtracellular

M

atrix

M

etallo

Pr

oteinase

In

ducer (EMMPRIN, originally des-ignated Tumor Collagenase Stimulatory Factor, TCSF), which induces stromal fibro-blasts to produce MMPs.

23, 24

In recent

in situ

hybridization studies of breast andlung cancer, EMMPRIN was identified in cancer cells, while adjacent stromal fibro-blasts produce gelatinase A and MT1-MMP. EMMPRIN has also been identified innormal epithelial cells (breast ductal cells, keratinocytes), suggesting similarities be-tween control of MMP synthesis in normal and malignant tissues.

Another important aspect of MMP and TIMP production in cancer is the questionof whether these proteins are produced early or late in the disease. Stromelysin-3 andmatrilysin are present early in tumor development (colon adenomas and breast car-cinoma

in situ

), at a time when tumors are not known to be invasive or metastatic;this observation suggests that stromelysin-3 and matrilysin may not be involved inmetastasis.

TISSUE MMP

S

AND TIMP

S

AS PROGNOSTIC MARKERS IN CANCER: CORRELATION WITH STAGE

Increased tumor tissue levels of gelatinase B, gelatinase A, collagenase-1, andMT1-MMP, identified by either mRNA measurement, bioassay, or immunohis-tochemistry, have been found to correlate with advanced cancer stage in gastrointes-tinal, breast, prostate, and bladder cancer.

10,14,25,26

Zeng

et al.

demonstrated byNorthern blot analysis that an increased ratio of tumor/normal mucosa gelatinase BmRNA correlated significantly with the status of distant metastasis and Dukes’ clin-ical staging for colorectal cancer.

27,28

The presence of activated gelatinase A and ge-latinase B in human cancer tissue extracts, as detected by substrate zymography, hasalso been proposed as a cancer marker in aggressive breast and colon adenocarcino-mas.

29,30

While Liabakk

et al.

likewise reported increased levels of gelatinase B andgelatinase A, as well as activated gelatinase A in colorectal cancer tissues, no corre-lation between gelatinase levels and survival was noted.

31

In a study of tumor spec-imens obtained from patients with various stages of colon cancer, Murnane

et al.

noted that activated gelatinase A appeared early and persisted, whereas gelatinase Bbecame more prominent during the advanced stages of colon cancer. Cathepsins Band L were elevated in early-stage cancer, but then decreased. These authors sug-gested that gelatinase A is involved in the initiation and maintenance of malignancyand gelatinase B is required for distant spread.

32

Nielsen

et al.

emphasized that neu-trophils and macrophages infiltrating colon

33

and breast cancer tissue are the majorsource of gelatinase B, with macrophage turnover of gelatinase B being rapid. Inter-action of T cells with macrophages via the gp39-CD40 counter receptors

34

is one po-tential mechanism for induction of macrophage gelatinase B. Increased levels ofTIMP-1 and TIMP-2 mRNA and protein have also been identified in malignant stro-mal tissues.

11,15,27,35

A correlation between TIMP-1 levels and Dukes’ stage of col-orectal cancer has been proposed.

15,35

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S

AND TIMP

S

Using specific monoclonal antibodies in immunoassays, Stearns reported thathigher levels of latent and activated gelatinase A in prostate tissue extracts correlatedwith more aggressive (higher Gleason score) and metastatic cancer.

36

Increased ge-latinase B secretion and a high ratio of gelatinase B to gelatinase A in short-term tis-sue culture was observed with prostate cancer as opposed to benign prostatichypertrophy.

37

Likewise, using

in situ

hybridization, high tissue expression of gelati-nase B and gelatinase A and low expression of TIMP-1 and TIMP-2 were indepen-dent predictors of poor outcome in prostate cancer.

6

Using reverse transcriptase PCRto measure gene expression in tissue obtained at surgery, Kanayama

et al.

reportedthat gelatinase A, TIMP-2, and MT1-MMP are useful prognostic indicators in pa-tients with bladder cancer. Patients with high expression of any of these three geneshad a worse prognosis than those with low expression after radical cystectomy.

38

In view of the fact that other types of proteinases (i.e., plasminogen activator,cathepsin B, cathepsin D) and proteinase inhibitors (i.e., plasminogen activator in-hibitor, calpain) have also been implicated in cancer metastasis, it is not surprisingthat the correlation between concentration of a single proteinase in tissues or bloodand clinical outcome is often disputed.

MEASUREMENT OF MMP

S

AND TIMP

S

IN BODY FLUIDS

Increased serum levels of the commonly used tumor markers (CEA, PSA,CA-125, HCG) provide useful information about the body tumor burden; these testshowever provide little information that is predictive of the biologic behavior of a par-ticular form of cancer. On the basis of detection of high levels of MMPs in humancancer tissues and the identification of MMPs in human plasma, we proposed thattissue MMPs may leach into the blood stream in increased amounts in patients withbiologically aggressive cancer and thereby provide unique markers that might beuseful for predicting metastasis. To explore this hypothesis, sandwich-type enzyme-linked immunosorbent assays (ELISA) have been developed to measure the concen-tration of GLB, GLB:TIMP-1 complexes, GLA, GLA:TIMP-2 complexes, andstromelysin-1 in the plasma of patients with cancer.

Numerous investigators have demonstrated that MMPs and TIMPs are readilymeasured in plasma and serum by ELISAs which employ specific polyclonal ormonoclonal antibodies. Mean levels of gelatinase A,

39

TIMP-1,

40

TIMP-2,

41

stromelysin-1,

40

gelatinase B, collagenase-1,

42

and matrilysin

43

in normal plasma/serum range from ~500 ng/ml to ~10 ng/ml (order of appearance reflects their rela-tive concentration). It should be remembered that the measurement of gelatinase Bin serum is unreliable, since it reflects, in large part,

in vitro

MMP release occurringduring degranulation of blood neutrophils;

44,45

plasma measurements are not con-founded by this artifact. One of the limitations of ELISAs is that the results achievedusing different antibodies and protein standards can vary, which complicates com-parison of results between various reports.

40,42

Another confounding factor affectingclinical MMP measurements is that MMPs are capable of binding to connective tis-sue matrix

46

; hence increased local secretion of MMPs in disease may not necessar-ily be translated into increased plasma levels. The degradation and excretion


pathways for MMPs and TIMPs in the body have not been examined to date. Thus,we can only assume that high blood levels of these proteins reflect increased produc-tion rather than diminished excretion.

In development of a prototype sandwich ELISA, Zucker

et al.

demonstrated thatplasma gelatinase A was significantly increased (>50%) in the second half of preg-nancy as compared to early pregnancy or the nonpregnant state.

39

In contrast to ex-pectations, plasma gelatinase A levels were not significantly increased in patientswith advanced gastrointestinal cancer, breast cancer, gynecologic cancer, lung can-cer, and lymphoma-leukemia as compared to levels in normal individuals. On the ba-sis of

in vitro

studies, it was proposed that endothelial cells and other normal cellsmake a sizable contribution to plasma levels of gelatinase A in the intact animal, andmay thereby obfuscate detection of increased levels of enzyme originating from sol-id tumors.

47

Fujimoto

et al.

likewise reported that serum gelatinase A levels were notincreased in stomach cancer and pancreatic cancer, but were increased (~30%) inhepatocellular carcinoma, hyperthyroidism, and biliary cirrhosis.

48

AlthoughGabrisa

et al.

49

reported increased serum gelatinase A in patients with metastaticlung cancer as compared to localized lung cancer, the differences were small.

In a study of patients with urothelial cancer, Gohji

et al

. demonstrated that the ra-tio of gelatinase A to TIMP-2 in serum was significantly higher in patients with re-current cancer. Disease-free survival in patients with high gelatinase A:TIMP-2ratios was poor as compared to patients with lower ratios; this ratio was shown to bean independent prognostic indicator of urinary tract cancer recurrence.

50

High serumstromelysin-1 and gelatinase A levels were also shown to be predictors for recurrentcancer in patients with advanced bladder cancer after complete tumor resection.

51

Incontrast to TIMP-2, serum levels of TIMP-1 were reported by Naruo

et al.

to be in-creased in patients with bladder cancer; increased TIMP-1 levels correlated withprogression of cancer as demonstrated by increased invasion and metastasis.

52

In-creased serum gelatinase A levels have been demonstrated in patients with prostatecancer; a correlation with clinical course in patients with bone metastasis was dem-onstrated.

53

Gelatinase B measurements in blood have provided more encouraging results incancer than have been achieved with the gelatinase A assay. Using a pair of mono-clonal antibodies to gelatinase B in a sandwich ELISA, Zucker

et al.

demonstratedthat gelatinase B was significantly increased in the plasma of patients with breastcancer and gastrointestinal tract cancer (metastatic and nonmetastatic) as comparedto normal subjects.

45

Likewise, latent gelatinase B:TIMP-1 complexes (TIMP com-plexes) were significantly increased in the plasma of patients with gastrointestinaltract cancer and female genitourinary tract cancer as compared to control subjects.Of interest, some patients had increased plasma levels of gelatinase B:TIMP-1 com-plexes and normal gelatinase B levels, and vice versa. When results from both thegelatinase B and the gelatinase B:TIMP-1 complex ELISAs were combined, in-creased levels of either gelatinase B, TIMP complexes, or both were found in 36%of patients with gastrointestinal cancer and 65% of patients with genitourinary tractcancer. Most importantly, clinical follow-up (40 months) of patients with stage IV(metastatic) gastrointestinal cancer indicated that the length of survival of patientswith increased plasma levels of gelatinase B or TIMP complexes was significantlyshorter than that of patients with normal plasma levels (4 months vs. 20 months, re-

217ZUCKER

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S

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S

spectively).

22

On the basis of these studies, Zucker

et al.

proposed that the assay ofgelatinase B and TIMP complexes in plasma may be clinically useful in predictingsurvival in certain subsets of cancer patients. An inverse relationship was identifiedbetween plasma gelatinase B and TIMP complexes versus CEA, suggesting that pro-duction of gelatinase B (presumably by tumor macrophages) occurs primarily in col-orectal cancers with low CEA production. It remains to be determined whethergelatinase B and TIMP complexes will be a marker for metastasis or prognosis inpatients with earlier stage colorectal cancer.

45

A practical limitation to these tests incancer patients undergoing treatment is that plasma gelatinase B levels have been re-ported to decrease in parallel with the drop in blood granulocytes in patients treatedwith chemotherapy.

44

An unsatisfying aspect of these studies is that we do not un-derstand the connection between the inflammatory cell infiltrates which are respon-sible for the production of high levels of gelatinase B and TIMP-1 and the poorprognosis in this subset of patients with colorectal cancer.

In these initial studies, even though significant differences exist between groups,the bimodal distribution of plasma MMPs and TIMP complexes in the normal pop-ulation limits the practical value of these assays for the individual patient since thereis considerable overlap in test results between the cancer patient population and thehealthy population.

39,44,45

Employing a more sensitive revised ELISA consisting of a polyclonal capture an-tibody and a monoclonal detecting antibody to gelatinase B, we have recently dem-onstrated that gelatinase B is increased in the plasma of more than 50% of patientswith breast cancer, prostate cancer, and bladder cancer, but not gynecologic cancers(ovary, cervix, uterus, vagina). In treated patients with prostate cancer, no correlationwas noted between the plasma concentration of gelatinase B and prostate-specificantigen (PSA), indicating that MMPs do not correlate with body tumor burden. Us-ing this ELISA, we have examined the effect of therapy on fluctuations of plasmagelatinase B in patients with breast cancer. Both chemotherapy and hormonal thera-py resulted in considerable fluctuations in plasma gelatinase B levels: in some in-stances there appeared to be a correlation with clinical progression/regression indisease activity, but not uniformly (F

IG

. 1). Elevated plasma levels of gelatinase Bhave also been reported in patients with hepatocellular carcinoma.

54

In a comprehensive study of serum MMPs and TIMPs, Baker

et al.

reported in-creased levels of TIMP-1 and collagenase-1 and lower levels of TIMP-2 in patientswith prostate cancer as compared to control subjects.

55

Patients with metastatic can-cer had significantly higher levels of collagenase-1 than did control subjects. Inagreement with other studies,

56

serum stromelysin-1 was not increased in patientswith cancer. Baker

et al.

emphasized that it is uncertain whether collagenase-1 andTIMP-1 are tumor-derived or result from tumor stromal cell expression in responseto tumor cell growth.

55

Jung

et al.

confirmed the finding of increased plasma levelsof TIMP-1 in prostate cancer, but also reported increased levels of stromelysin-1 inprostate cancer patients with metastases.

57

On the basis of our studies in rheumatoiddisease,

56,58

we suspect that the minimal increase in stromelysin-1 in prostate cancerreflects an inflammatory type of response rather than a cancer effect.

We have recently developed an ELISA for measurement of matrilysin (MMP-7)using a rabbit polyclonal antibody to recombinant antigen as the capture antibody


and a rat monoclonal antibody as the detecting antibody. In preliminary studies, itbecame obvious that a bimodal distribution of matrilysin values was noted within acontrol population of men and women; healthy people had either plasma matrilysinlevels <5 ng/ml or >100 ng/ml. The comparable bimodal distribution of matrilysinlevels was also noted in macrophage-conditioned media collected from healthy vol-unteers. Measurement of matrilysin in cancer patients demonstrated increased plas-ma levels only in patients with bladder cancer, but not in breast, GI, lung,gynecologic, or prostate cancer (F

IG

. 2). These data are contrary to expectation sincehigh levels of matrilysin have been identified in pathologic tissues and cell lines de-rived from patients with GI, breast, and prostate cancer.

59

Of interest, gelatin zymography has recently been used to examine the frequencyof detection of MMPs in the urine of patients with cancer. Gelatinase A and gelati-nase B were demonstrated in the urine of most patients with active breast, bladder,and prostate cancer, but infrequently in patients with cancer and no evidence of dis-ease or in healthy control subjects. An unclassified high molecular weight species ofgelatinase (125–150 kDa) was also demonstrated in the urine of cancer patients.

60

FIGURE 1. Line chart showing the fluctuation of plasma gelatinase B (measured byELISA) in a 57-year-old woman with breast cancer who was undergoing chemotherapy andhormonal therapy. This patient had invasive ductal carcinoma of the breast with a large ax-illary lymph node metastasis. A mastectomy had been performed one year earlier and wasfollowed by adjuvant chemotherapy. The cancer recurred (®) (listed as month zero), and thepatient was treated with Taxol for 4 months. Skull metastases were then noted, and tamox-ifen was initiated. New metastatic lesions were noted at 12 months, and aminoglutethemidewas started. Disease progression at 16 months was followed again by a rise in plasma ge-latinase B. The patient died at 20 months after operation.

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S

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S

STROMELYSIN-1 LEVELS IN BLOOD OF PATIENTSWITH INFLAMMATORY DISEASE

Numerous reports described increased levels of serum/plasma stromelysin-1 lev-els in patients with rheumatoid arthritis and to a lesser degree in patients with os-teoarthritis and gout. Approximately 80% of patients with rheumatoid arthritis had3-fold increased blood levels of stromelysin-1; higher serum levels of stromelysin-1correlated with certain parameters of disease activity.

55,56,61–63

Synovial fluidstromelysin-1 levels are several hundred-fold higher than serum levels, which is con-sistent with local stromelysin-1 production in the joint and subsequent leaching intothe blood stream. In arthritis, an excellent correlation between paired serum and syn-ovial fluid levels of stromelysin-1 suggests that serum levels accurately reflect intra-articular events.

61

Serum collagenase-1 is increased to a lesser degree than isstromelysin-1 in patients with arthritis.

Elevated serum stromelysin-1 levels in inflammatory bowel disease

64

and graft-versus-host disease suggests that mesenchymal cell production of stromelysin-1 isunder the control of numerous cytokines (i.e., TNF, IL-1), thereby limiting the use-fulness of stromelysin-1 measurements in differential diagnosis of inflammatory dis-eases.

FIGURE 2. Point chart showing the mean ± standard error of plasma matrilysin in nor-mal individuals, pregnant women, and patients with prostate, bladder, breast, GI, gyneco-logic, and lung cancers. Plasma matrilysin was measured by ELISA. Bladder cancer was theonly group in which plasma levels were significantly above those of patients in the normalgroup (as indicated by the asterisk).


Systemic lupus erythematosus, the prototype autoimmune disease, is another dis-ease in which we might anticipate enhanced MMP production. In a recent study byZucker et al., serum MMP levels and SLE Disease Activity Indices were measuredin a large number of patients with SLE and in healthy controls. Results indicated a3–5-fold increase in serum stromelysin-1 levels in patients with SLE as compared tohealthy subjects. Contrary to expectations, serial measurements of stromelysin-1 inindividual patients with SLE did not correlate with fluctuation in disease activityscores. Collagenase-1, gelatinase A, and TIMP-1 levels were not significantly in-creased in the serum of patients with SLE as compared to control subjects.58 Thesedata suggested that stromelysin-1 may not be primarily involved in the initial tissuedamage in SLE, but rather participates in a later aspect of inflammation involvingtissue repair. If this theory is correct, the use of MMP-inhibiting drugs may prove tobe detrimental in diseases in which the MMP repair mechanism is beneficial to theorganism. In these instances, MMP inhibitory drugs may be useful early in disease,when the destructive aspects of MMPs are prominent, but not during the later repairstages of disease. The importance of clinical trials to clarify these issues cannot beunderestimated.

SERUM MMPS AND TIMPS IN LIVER DISEASE

In contrast to cancer and arthritis, many disease processes are associated with in-creased extracellular matrix deposition rather than degradation. The hallmark of tis-sue injury in response to chronic excess alcohol consumption is increased hepaticfibrosis (cirrhosis). Collagen accumulation reflects not only enhanced synthesis, butalso results from a failure of collagen degradation to keep pace with collagen produc-tion. Several groups have demonstrated increased serum and liver tissue levels ofTIMP-1 in patients with alcoholic cirrhosis associated with liver fibrosis.65 SerumTIMP-1 levels correlated more closely with the histologic degree of liver fibrosis thanwith hepatic inflammation or necrosis. Collagenase-1, gelatinase A, gelatinase B, andstromelysin-1 are also produced by the liver in response to injury. It is well estab-lished that collagenase-1 activity is decreased as the fibrosis progresses in cirrhoticlivers.66 A recent study has demonstrated that patients with hemochromatosis and he-patic fibrosis have increased ratios of TIMP-1 compared to either collagenase-1, ge-latinase A, or stromelysin-1.67 In patients with chronic hepatitis C treated withinterferon, responders had an increase in serum collagenase-1 and a decrease in se-rum TIMP-1; nonresponders had the reverse effect, suggesting that interferon mayexert a beneficial effect on hepatic fibrosis.68

SERUM MMPS AND TIMPS IN VASCULAR DISEASE

There is considerable current interest in the role of MMPs in cardiovascular dis-ease, especially in regard to the pathogenesis of dissecting aortic aneurysms and rup-ture of atherosclerotic plaques.69 Of interest, Hirohata et al. have described a dropin serum collagenase-1 and TIMP-1 persisting for several days after acute myocar-dial infarction and then a return to normal levels. A correlation with cardiac functionwas noted suggesting the involvement of collagenase in the healing process duringcardiac remodeling.70

221ZUCKER et al.: MEASUREMENT OF MMPS AND TIMPS

PLASMA MMPS AND TIMPS IN PREGNANCY

It is well established that MMP function is important in many aspects of fetal im-plantation, pregnancy, and delivery. In a recent study, Tu et al. demonstrated thatplasma gelatinase B levels are more than 15-fold increased from week 19 to 36 ofpregnancy. Plasma gelatinase B levels during spontaneous labor increased an addi-tional 3-fold regardless of gestational age.71 The tissue origin of gelatinase B inpregnancy is presumed to be the choriodecidual membranes. In contrast, Kolben etal.72 reported that plasma concentrations of gelatinase B rise between the 10th and40th week of pregnancy. A role for IL-8 in increasing the release of gelatinase B andcollagenase-2, resulting in cervical ripening during labor, has been proposed.73 Asimilar pattern of gestational plasma levels is seen for collagenase-1, except that theprenatal levels do not differ from those of nongravid controls, but become dramati-cally elevated at term labor.74 In uncomplicated pregnancies, serum TIMP-1 levelswere low during pregnancy until the 37th week, when the levels rose to that of thenonpregnant state. Serum TIMP-1 levels rose during labor and the postpartum peri-od. The contrast between changes of MMPs and TIMP during pregnancy serve to re-emphasize the balance/imbalance occurring during various physiologic states.75

MEASUREMENT OF MMP AND TIMP IN BRONCHOALVEOLAR LAVAGE FLUID (BAL)

Patients with serious lung disease frequently are subjected to bronchoscopy fordiagnostic or therapeutic purposes. We have examined BAL samples from 30 pa-tients with infectious, inflammatory, and malignant lung disease to determine wheth-er the concentration of MMPs and TIMPs reflect the underlying disease process.Gelatin zymography and ELISAs were performed. The results indicated that: (1) ge-latinase B is the dominant MMP in BAL (605 ± 159 pM); (2) the mean concentra-tions of gelatinase A (24 ± 1 pM), collagenase-1 (93 ± 28 pM), and stromelysin-1(80 ± 7 pM) were considerably lower; (3) the concentration of TIMP-2 (724 ± 81pM) exceeded that of TIMP-1 (284 ± 156 pM); (4) only modest differences were not-ed between levels of MMPs and TIMPs in these patients; and (5) patients with asth-ma had significantly higher levels of gelatinase B (2454 ± 1454 pM) and TIMP-2than any of the other groups, including lung cancer (FIG. 3). By contrast to theseBAL results, the normal plasma levels of gelatinase A and gelatinase B are~7000 pM and 100 pM, respectively. This reversal of the relative concentration ofthese two MMPs in BAL as contrasted with normal plasma suggests that the gelati-nase B in BAL is not simply a transudate of plasma, but rather is produced locally inthe lung, primarily by macrophages lining the bronchi and alveoli. Furthermore,these data confirm that local MMP synthesis is often increased in nonmalignant dis-eases equivalent to malignancy. This has an impact on the interpretation of plasmaMMP measurements in differential diagnosis.


FIGURE 3B. Concentrations of TIMP-2 (pM) in BAL samples from five clinicalgroups with means ± SEM also shown. Significant differences (p<0.05) are shown (*).Asthmatic patients had the most elevated levels.

FIGURE 3A. Gelatinase B concentrations (pM) from BAL samples in each of five clin-ical groups with means ± SEM. Gelatinase B concentrations in patients with lung cancer andCOPD are more tightly grouped around the mean. Patients with asthma had significantlyhigher gelatinase B than did each of the other groups.


PROSPECTS FOR THE FUTURE

In conclusion, we propose that the assay of metalloproteinases and their complex-es with TIMPs in the plasma of patients with cancer may be clinically useful in char-acterizing the metastatic potential of certain types of cancer. These measurementswill also be useful in nonmalignant diseases characterized by excess tissue degrada-tion. From a theoretical point of view, the detection of activated gelatinases or com-plexes of activated gelatinase with the noncorresponding TIMP (gelatinase A:TIMP-1 complex or gelatinase B:TIMP-2 complex) may provide a better plasma testfor monitoring metastasis in patients with cancer than the assay of latent enzymesand latent MMP:TIMP complexes since activation of MMPs is ultimately requiredfor matrix degradation. Improvement in the sensitivity of these “complex” ELISAswill be required before their full potential can be recognized, since the concentrationof activated gelatinase complexes in plasma appears to make up less than 1% of thelevel of the latent enzymes.

ACKNOWLEDGMENTS

This research was supported by Grant No. DAMD 17-95-1 from the Unites StatesArmy Medical Research and Development Command, a VA Merit Review Grant,and a Carol Baldwin Cancer Grant (SUNY at Stony Brook).

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