ARTHRITIS & RHEUMATOLOGY Vol. 66, No. 3, March 2014, pp 726–730 DOI 10.1002/art.38277 © 2014, American College of Rheumatology
BRIEF REPORT
Lysyl Oxidase Is a Potential Biomarker of Fibrosis in Systemic Sclerosis Doron Rimar,1 Itzhak Rosner,1 Yuval Nov,2 Gleb Slobodin,1 Michael Rozenbaum,1 Katy Halasz,1 Tharwat Haj,1 Nizar Jiries,1 Lisa Kaly,1 Nina Boulman,1 Rula Daood,1 and Zahava Vadasz1 high serum LOX levels in SSc patients that correlate specifically with skin fibrosis. These correlations suggest that LOX levels may serve as a novel biomarker of fibrosis. Future studies are warranted to determine whether LOX is a potential therapeutic target in SSc.
Objective. Fibrosis is a major cause of morbidity and mortality in systemic sclerosis (SSc). Levels of lysyl oxidase (LOX), an extracellular enzyme that stabilizes collagen fibrils, have been found to be elevated in the skin of SSc patients, but have not been evaluated in the serum or correlated with the clinical parameters. We undertook this study to evaluate serum LOX levels in SSc patients and to correlate these levels with clinical parameters of SSc. Methods. SSc patients were evaluated for demographic features, clinical manifestations, routine laboratory tests, serum autoantibodies, serum LOX concentrations, and nailfold capillaroscopy patterns. They underwent pulmonary function testing, echocardiography, and high-resolution computed tomography scans of the lung, assessment of skin fibrosis by the modified Rodnan skin thickness score (MRSS), and assessment of disease severity and activity by the Medsger severity scale and the Valentini activity index. Results. Twenty-six SSc patients were evaluated and compared with 25 healthy controls and with 9 disease control patients with primary myelofibrosis. Almost 62% of the SSc patients (16 of 26) had limited cutaneous SSc (lcSSc), while 38% had diffuse cutaneous SSc (dcSSc) (10 of 26); 31% of the patients (8 of 26) had lung involvement. The LOX concentration in SSc patients was higher than that in healthy controls and similar to that in disease controls (P < 0.0001), and it was significantly higher in patients with dcSSc than in those with lcSSc (P ⴝ 0.006). The LOX concentration correlated with the MRSS in patients without lung fibrosis. Conclusion. This study is the first to demonstrate
Fibrosis is the main complication of systemic sclerosis (SSc) and its pathophysiology is complex. Despite our growing understanding of this process and the many targets available, our therapeutic success in ameliorating fibrosis in SSc is minimal (1). Moreover, even today, assessment of skin fibrosis is usually determined by the modified Rodnan skin thickness score (MRSS) (2), which is based on clinical inspection of 17 parts of the body, has significant interobserver variability, and is rather subjective (3). This is why other objective and specific markers for assessing fibrosis are needed. Type I collagen is the most abundant structural protein of the connective tissues, such as the skin. The formation of collagen is an active process that reflects a balance between degradation and synthesis and involves bone morphogenetic protein 1 (BMP-1), a disintegrin and metalloproteinase. Under physiologic conditions, the chemical crosslinking of collagen molecules incorporated in collagen fibrils is critical for the mechanical stability of these fibrils. Moreover, the presence of chemical crosslinks makes fibril-incorporated collagen molecules more resistant to proteolysis. Formation of crosslinks is an enzymatic process catalyzed by lysyl oxidase (LOX) (4). LOX is a copper-dependent amine oxidase that initiates the covalent crosslinking of collagen and elastin by catalyzing oxidative deamination of lysine and hydroxylysine residues to aminoadipic semialdehydes. These highly reactive semialdehydes can spontaneously condense to form intramolecular and intermolecular covalent crosslinkages that assure extracellular matrix stability. LOX activity is essential to maintaining the tensile and elastic features of the connective tissues of the skeletal, pulmonary, and cardiovascular systems,
1 Doron Rimar, MD, Itzhak Rosner, MD, Gleb Slobodin, MD, Michael Rozenbaum, MD, Katy Halasz, Tharwat Haj, PhD, Nizar Jiries, MD, Lisa Kaly, MD, Nina Boulman, MD, Rula Daood, MD, Zahava Vadasz, MD, PhD: Bnai Zion Medical Center, Haifa, Israel; 2 Yuval Nov, PhD: University of Haifa, Haifa, Israel. Address correspondence to Zahava Vadasz, MD, PhD, Division of Allergy and Clinical Immunology, Bnai Zion Medical Center, PO Box 4940, Haifa 31048, Israel. E-mail:
[email protected]. Submitted for publication May 1, 2013; accepted in revised form November 7, 2013.
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among others. LOX is synthesized as a preproLOX and is secreted into the extracellular environment, where it is proteolytically processed by BMP-1 and other procollagen C-proteinases to release the mature and active 32-kd form and its propeptide (5). LOX has been evaluated in patients with diffuse cutaneous SSc (dcSSc) by immune staining of the skin and was found to be increased in interstitial fibroblastic cells as compared with normal skin, but was not increased in SSc patients with skin atrophy (6,7). LOX has been evaluated in other fibrotic conditions, including primary myelofibrosis (8), hepatic fibrosis (9), and myocardial fibrosis (5), and was found to be overexpressed in the relevant tissues. Primary myelofibrosis is a myeloproliferative disorder, the manifestations of which include anemia, extramedullary hematopoiesis, splenomegaly, and typically intense bone marrow fibrosis with early mobilization of hematopoietic stem cells. Recently, high levels of LOX were found in bone biopsy samples and serum from patients with primary myelofibrosis as compared to controls (8). There are currently no data concerning serum LOX levels in SSc patients, nor have there been any assessments to date of the potential of LOX as a biomarker. Therefore, we conducted this study to evaluate serum LOX levels in SSc patients compared to normal controls and patients with primary myelofibrosis (disease controls), and we correlated these levels with clinical parameters. PATIENTS AND METHODS Patient population. Three groups of patients were enrolled in this study. Twenty-six consecutive patients who fulfilled the American College of Rheumatology classification criteria for SSc (10) were recruited from the Bnai Zion Medical Center rheumatology clinics and ambulatory care facility. Twenty-five age- and sex-matched individuals from the hospital staff were recruited as healthy controls. Nine patients with primary myelofibrosis diagnosed by bone marrow biopsy were recruited from the hematology clinic and served as the disease control group. Demographic characteristics and clinical manifestations of the SSc patients were assessed by history (age, sex, weight, height, smoking history, dyspepsia, and diarrhea), physical examination (evaluation of skin involvement as limited cutaneous SSc [lcSSc] or dcSSc according to the criteria of LeRoy et al [11], the MRSS, current digital ulcer, fissures, telangiectasia), and medical records (duration of disease, calcinosis, myositis, gastritis, past digital ulcers and critical ischemia, echocardiography and lung function testing in the last year, chest radiography, computed tomography [CT] scan, and medical treatment). Lung fibrosis was diagnosed if ⬎20% of the lung showed fibrotic changes on high-resolution CT scan. Past diagnosis of lung fibrosis was validated by reviewing the most recent lung CT scan. In patients not previously
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diagnosed as having lung disease, a CT scan was ordered if pulmonary fibrosis was suspected by lung auscultation (10 points), abnormal finding on chest radiograph, a diffusing capacity for carbon monoxide ⬍80% predicted, or forced vital capacity (FVC) ⬍80% predicted. Laboratory assessments included a complete blood cell count, levels of creatinine, urea, autoantibodies (anticentromere, anti–Scl-70, antineutrophil cytoplasmic antibody), C3, C4, and C-reactive protein erythrocyte sedimentation rate, and spot urine test or 24-hour urine collection for protein. Each patient underwent nailfold videocapillaroscopy, with findings classified as early (well-preserved capillary distribution, few giant capillaries and hemorrhages), active (4–6 capillaries per field and giant capillaries and hemorrhages in most fields), or late (less than 4 capillaries per field and neoangiogenesis) (12). Disease severity and activity were determined by the Medsger severity scale (13) and the Valentini activity index (13) during the visit and were subsequently reevaluated after further laboratory and imaging studies (14). Exclusion criteria were pregnancy, age ⬍18 years, other coexisting connective tissue disease, and other known fibrotic states. The study was approved by the local Research Ethics Board, and all patients gave their informed consent. Measurement of serum LOX. Serum LOX concentrations were determined with a commercial enzyme-linked immunosorbent assay kit according to the instructions of the manufacturer (USCN). Statistical analysis. Continuous data are presented as the mean ⫾ SD. Categorical variables are presented as frequencies and percentages. Since LOX concentrations were found not to have a Gaussian distribution, we log-transformed the data. Comparisons between categories were made using 2-tailed t-tests for continuous variables and the chi-square test for categorical variables. We further evaluated relationships between the LOX level (log-transformed) and disease-related covariates. For numerical covariates, the relationship was studied by a correlation test and is reported via Pearson’s r. For binary covariates, the relationship was studied by a t-test and is reported as the difference between means of the logtransformed data and the difference between means of the raw data (Table 1). Statistical analysis was performed using SPSS software (PASW Statistics 17.0.2, 2009).
RESULTS Patients. Twenty-six patients with SSc, 9 patients with primary myelofibrosis, and 25 healthy controls were enrolled in the study. There were no significant differences between the groups with respect to their age, ethnic origin, body mass index (BMI), and smoking habits. Table 2 summarizes the demographic and clinical (disease progression, immunologic data, and treatment) characteristics of the SSc patients along with the demographic characteristics and BMI of the other 2 groups. As shown in Table 2, the percentage of females was smaller in the group of patients with primary myelofibrosis than in the other 2 groups. Almost 62% of SSc
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Table 1. Relationship between the lysyl oxidase level and diseaserelated covariates in the cohort of SSc patients (n ⫽ 26)*
Covariate Age, years Female sex BMI, kg/m2 Current smoker Past smoker Years since diagnosis of non-RP manifestation lcSSc DLCO, % predicted FVC, % predicted Systolic PAP, mm Hg MRSS Active digital ulcer in past 3 months History of critical ischemia Late nailfold videocapillaroscopy pattern Diastolic or systolic dysfunction Diarrhea Calcinosis Myositis ACA Anti–Scl-70 Valentini activity score Treatment Iloprost Bosentan Sildenafil ACE inhibitors CCB Methotrexate Azathioprine Cyclophosphamide, ever Cyclosporine Rituximab Mycophenolate mofetil IVIG
r or difference between means of log-transformed data (difference between means of raw data)† –0.002 0.566 (28.28) 0.275 –0.1649 (–11.54) –0.199 (–10.16) ⫺0.146 –0.439 (–23.87) 0.045 –0.097 –0.19 0.374 –0.109 (–5.71) 0.270 (18.87) 0.153 (12.36) 0.240 (16.71) 0.178 (12.44) 0.029 (7.65) –0.092 (–6.20) –0.0593 (–2.62) 0.337 (18.92) 0.246 –0.0317 (–2.59) 0.101 (7.85) 0.462 (27.80) 0.281 (16.35) 0.2363 (14.90) 0.1628 (10.56) 0.0995 (8.79) 0.316 (15.84) –0.0770 (–3.91) –0.237 (–14.26) 0.740 (54.66) –0.141 (–8.53)
* The only significant differences were for limited cutaneous systemic sclerosis (lcSSc) (P ⫽ 0.003) and the modified Rodnan skin thickness score (MRSS) (P ⫽ 0.0596). BMI ⫽ body mass index; RP ⫽ Raynaud’s phenomenon; DLCO ⫽ diffusing capacity for carbon monoxide; FVC ⫽ forced vital capacity; PAP ⫽ pulmonary artery pressure; ACA ⫽ anticentromere antibody; ACE ⫽ angiotensin-converting enzyme; CCB ⫽ calcium-channel blocker; IVIG ⫽ intravenous immunoglobulin. † For numerical covariates, the relationship was accessed by correlation test and is reported via Pearson’s r. For binary covariates, the relationship was accessed by t-test and is reported as the difference between means of the log-transformed data (difference between means of the raw data).
patients (16 of 26) had lcSSc, while ⬎38% had dcSSc (10 of 26); 31% of the SSc patients had lung involvement (8 of 26). Serum LOX levels. As shown in Figure 1A, the mean ⫾ SD log-transformed serum LOX levels (and serum LOX levels) in SSc patients were higher than those in healthy controls but similar to those in patients
with primary myelofibrosis (1.73 ⫾ 0.18 [58.4 ⫾ 4.8 ng/ml] versus 1.38 ⫾ 0.25 [28.4 ⫾ 2.5 ng/ml] versus 1.58 ⫾ 0.25 [44.6 ⫾ 9.4 ng/ml], respectively; P ⬍ 0.0001). Moreover, the mean ⫾ SD log-transformed serum LOX Table 2. Demographic and clinical characteristics of the study population*
Variable Age, mean ⫾ SD years Female sex BMI, mean ⫾ SD kg/m2 Jewish origin Current smoker Past smoker Years since diagnosis of non-RP manifestation, mean ⫾ SD Lung fibrosis dcSSc lcSSc DLCO, mean ⫾ SD % predicted FVC, mean ⫾ SD % predicted Systolic PAP, mean ⫾ SD mm Hg MRSS, mean ⫾ SD Active digital ulcer in past 3 months History of critical ischemia Capillaroscopy pattern Early Active Late Diastolic or systolic dysfunction Diarrhea Calcinosis Myositis SRC ACA Anti–Scl-70 ANA Proteinuria Valentini activity score, mean ⫾ SD Medsger severity score, mean ⫾ SD Treatment Iloprost Bosentan Sildenafil ACE inhibitors CCB Methotrexate Azathioprine Cyclophosphamide, ever Cyclosporine Rituximab Mycophenolate mofetil IVIG
SSc patients (n ⫽ 26)
Patients Healthy with PMF controls (n ⫽ 9) (n ⫽ 25)
48.4 ⫾ 12.6 24 (92) 24 ⫾ 3.7 22 (84) 2 (7.7) 7 (27) 5.9 ⫾ 4.6
55 ⫾ 4.9 46.1 ⫾ 3.9 5 (55) 24 (96) 26 ⫾ 5.2 25 ⫾ 4.6 7 (77) 21 (84) 1 (11.1) 2 (8) 3 (33) 6 (24) – –
8 (31) 10 (38.5) 16 (61.5) 69 ⫾ 29 85.9 ⫾ 20.8 29.8 ⫾ 8.9
– – – – – –
– – – – – –
14.6 ⫾ 11.3 15 (58)
– –
– –
7 (27)
–
–
5 (19) 7 (27) 14 (54) 6 (23) 6 (23) 17 (65) 4 (15) 0 (0) 11 (42) 7 (27) 26 (100) 3 (11.5) 2.5 ⫾ 1.7
– – – – – – – – – – – – –
– – – – – – – – – – – – –
5.5 ⫾ 3.2
–
–
17 (65) 16 (61) 2 (8) 5 (19) 7 (27) 16 (61) 6 (23) 8 (31) 8 (31) 2 (8) 1 (4) 4 (15)
– – – – – – – – – – – –
– – – – – – – – – – – –
* Except where indicated otherwise, values are the number (%). The only significant difference was the number of females in the group of patients with primary myelofibrosis (PMF) (P ⫽ 0.045 versus the other 2 groups). dcSSc ⫽ diffuse cutaneous SSc; SRC ⫽ scleroderma renal crisis; ANA ⫽ antinuclear antibody (see Table 1 for other definitions).
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Figure 1. A, Comparison of serum lysyl oxidase (LOX) concentrations between systemic sclerosis (SSc) patients, patients with primary myelofibrosis (PMF), and healthy controls (CON). B, Comparison of serum LOX concentrations between SSc patients with limited disease and those with diffuse disease. C, Linear regression between LOX concentration and modified Rodnan skin thickness score (MRSS) in SSc patients. Values in A and B are the mean ⫾ SD. Dashed lines in C represent upper and lower confidence limits of the regression line.
levels (and serum LOX levels) were higher in patients with dcSSc than in those with lcSSc (1.85 ⫾ 0.12 [73 ⫾ 6.6 ng/ml] versus 1.66 ⫾ 0.76 [49.3 ⫾ 5.5 ng/ml]; P ⫽ 0.006) (Figure 1B). Clinical correlations. In a univariate analysis, log-transformed serum LOX levels showed a trend toward correlation with the MRSS (P ⫽ 0.0596). A further analysis of a subgroup of patients without lung fibrosis (n ⫽ 16) revealed an even stronger correlation of log-transformed serum LOX levels with the MRSS (r ⫽ 0.58, P ⫽ 0.0) (data not shown). DISCUSSION The present study has demonstrated that serum LOX levels are elevated in patients with SSc compared to those in healthy controls and are similar to those in control patients with primary myelofibrosis. Moreover, LOX levels were higher in SSc patients with dcSSc (most of them with lung fibrosis) than in those with lcSSc. Further analysis revealed a correlation of LOX concentration with the MRSS in patients without lung fibrosis. In SSc, there are 2 main sites of fibrosis (i.e., the skin and the lung). Former studies that found LOX to be overexpressed in skin fibroblasts of patients with dcSSc are consistent with our finding of a correlation between the peripheral blood LOX level and the MRSS, a measure of skin involvement (9,12). That the LOX level was higher in patients with dcSSc (most of whom also had lung fibrosis) than in those with lcSSc is not surprising and may reflect advanced fibrosis at both sites. FVC is reduced in patients with fibrotic lung disease; therefore, it would be expected to be inversely correlated with LOX levels. Yet this correlation did not reach statistical significance. This may be related to the small sample size and to the nature of the test, which depends on the cooperation of the patient. Indeed, 2 patients with lcSSc and low serum LOX levels had
reduced FVC but no evidence of interstitial lung disease on lung CT scan. The fact that LOX levels did not correlate with factors related to vasculopathy or autoantibodies in SSc is consistent with a specific relationship of LOX solely with fibrosis. At present, several biologic processes critical for the development of fibrotic lesions have been considered potential targets for inhibitors of fibrosis. These inhibitors aim at reducing inflammatory processes, inhibiting biologic functions of cytokines, reducing cell proliferation, and decreasing the biosynthesis and enzymatic processing of procollagen molecules (15). The above approaches target broad upstream processes, and therefore, interfering with them is frequently associated with adverse effects and has not proved successful as yet (1). Since it is located downstream in this process, LOX is a potentially interesting therapeutic target. LOX enzymatic activity is inhibited irreversibly by -aminopropionitrile (-APN), which has been used in animal models in the context of tissue fibrosis. In a study of 10 patients with scleroderma, -APN was not found to be effective mostly because of severe side effects, and the treatment had to be withdrawn shortly after initiation (16). Another drug that may have to do with LOX activity is D-penicillamine, which was once considered a potent antifibrotic drug (17). D-penicillamine is a strong chelator of copper, which is crucial for the activation of LOX, and hence, LOX inactivation may be at least part of the mode of action of penicillamine in SSc. Finally, a humanized antibody to LOX-like 2, a member of the LOX family, is currently being evaluated as a treatment for liver fibrosis (http://clinicaltrials.gov/ ct2/show/NCT01672879?term⫽lysyl⫹oxidase&rank⫽4). A humanized anti-LOX antibody may be a potential antifibrotic drug in SSc. Our study had several limitations, the most important of which is the small sample size, which may
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have affected the accuracy of the statistical analysis. Noteworthy is the lower percentage of women in the disease control group compared to the other groups. Since LOX levels were not correlated with sex and were actually lower in men with primary myelofibrosis than in women with primary myelofibrosis, it seems that this sex bias was not responsible for the higher LOX levels than those in healthy controls. Our study population was heavily treated with biologic and nonbiologic drugs, which may have had an effect on the results. Finally, we evaluated every patient only once, and therefore, we do not know if the value of LOX is constant or is altered according to treatment protocol or disease progression, a question that should be answered in ongoing studies. In summary, we have found LOX to be a novel biomarker that correlates with the MRSS in patients with SSc without lung fibrosis. We suggest that this correlation may reflect the extent of fibrosis in these patients. Future studies are needed to validate our findings and to consider LOX as a potential target for antifibrotic treatment.
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AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Vadasz had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Rimar, Rosner, Nov, Slobodin, Rozenbaum, Haj, Jiries, Kaly, Boulman, Daood, Vadasz. Acquisition of data. Rimar, Rosner, Nov, Slobodin, Rozenbaum, Halasz, Jiries, Kaly, Boulman, Daood. Analysis and interpretation of data. Rimar, Rosner, Nov, Halasz, Haj, Vadasz.
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