Analytical methods for measuring collagen XIX in human cell cultures, tissue extracts, and biological fluids

Analytical Biochemistry 437 (2013) 111–117

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Analytical methods for measuring collagen XIX in human cell cultures, tissue extracts, and biological fluids J.B. Oudart a,b,1, S. Brassart-Pasco a,1, E. Luczka a, A. Dupont-Deshorgue a, G. Bellon a,b, S.P. Boudko c, H.P. Bächinger c, J.C. Monboisse a,b, F.X. Maquart a,b, L. Ramont a,b,⇑ a b c

Université de Reims Champagne-Ardenne, FRE CNRS No. 3481, Reims, France CHU de Reims, Laboratoire Central de Biochimie, Reims, France Research Department, Shriners Hospital for Children, Portland, OR 97239, USA

a r t i c l e

i n f o

Article history: Received 27 December 2012 Received in revised form 4 March 2013 Accepted 5 March 2013 Available online 14 March 2013 Keywords: Collagen XIX Western blot Real-time PCR Competitive ELISA

a b s t r a c t Type XIX collagen is a minor collagen associated with basement membranes in vascular, neuronal, mesenchymal, and epithelial tissues. We demonstrated that the NC1, C-terminal, domain of collagen XIX inhibits the migration capacities of tumor cells and exerts a strong inhibition of tumor growth. Other basement membrane collagens or derived fragments were measured in biological fluids such as blood and urine of patients and appeared to be useful for diagnosis, prognosis, or treatment monitoring. The aim of this study was to develop and validate methods to measure collagen XIX and its fragments in human cell cultures, tissue extracts, and human biological fluids. For that purpose, we developed realtime PCR, Western blot, and competitive enzyme-linked immunosorbent assays. We demonstrated that the methods developed in this paper are specific for collagen XIX. We showed that it is expressed in human cell cultures, tissue extracts, and various biological fluids. These methods may be used in various human tissue extracts and biological fluids such as serum, amniotic fluid, cord blood, and many other fluids. Collagen XIX or its fragments could constitute new biomarkers for human diseases as well as for diagnosis and/or prognosis. Ó 2013 Elsevier Inc. All rights reserved.

The basement membrane is a complex structure [1], long regarded as a simple cell support. However, it exerts many important biological functions, well described today, and its roles in many physiological and pathological situations are now well established [2]. Measurement of several basement membrane components are used as diagnostic and prognostic markers in many diseases, and some of them are used as tumor markers. For example, measurements of type IV collagen, the main component of the basement membranes, or of type XVIII collagen, a minor component of basement membranes, were used as tumor markers [3,4]. Enzyme-linked immunosorbent assays (ELISA)2 have been developed to measure fragments of these collagens in various human biological fluids [5]. Type XIX collagen is a minor collagen associated with basement membranes. It is a homotrimer of 400 kDa, composed by the association of three a1(XIX) chains. Each chain comprises 1142 residues, including six noncollagenous domains (NC1-NC6) separated by five collagenous domains (Col1-Col5) [6,7]. It is localized in ⇑ Corresponding author. Address: Laboratoire Central de Biochimie, CHU de Reims, Rue Serge Kochman, 51092 Reims Cedex, France. Fax: +33 326788539. E-mail address: [email protected] (L. Ramont). 1 These two authors contributed equally to the study. 2 Abbreviations used: BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assays; PCR, polymerase chain reaction; PMSF, phenylmethylsulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2013.03.007

the basement membrane zone of vascular, neuronal, mesenchymal, and epithelial tissues, associated with type IV or type XVIII collagens [8]. Collagen XIX is ubiquitously expressed during embryogenesis, where it participates in extracellular matrix assembly. During breast cancer progression, collagen XIX disappears from basement membrane at invasive stages [9]. Previously, we demonstrated that the NC1, C-terminal, domain of collagen XIX exerts a strong inhibition of tumor growth in an experimental murine melanoma model [10]. In vitro, NC1(XIX) also inhibits the migration capacities of tumor cells [11]. The aim of this study was to develop and validate methods for measuring collagen XIX expression. These methods can be used to investigate the expression of collagen XIX in cell cultures, in human fluids, or in tissue extracts. For that purpose, we developed a real-time PCR assay as well as Western blot and competitive enzyme-linked immunosorbent assays. Materials and methods Reagents Culture media and molecular biology products were from Life Technologies (Invitrogen, Strasbourg, France). All other reagents were purchased from Sigma (St. Quentin-Fallavier, France). Rabbit

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anti-human NC1 a1(XIX) was produced by Covalab (Villeurbanne, France). The NC1(XIX) peptide, NPEDCLYPVSHAHQRTGGN, was purchased from Proteogenix (Oberhausbergen, France). It was obtained by solid-phase synthesis using a FMOC (N-(9-fluorenyl)methoxy-carbonyl) derivative procedure and further purified by reverse-phase high-performance liquid chromatography with a C18 column eluted by a gradient of acetonitrile in trifluoroacetic acid, and then lyophilized [12]. A trimeric mini collagen containing 6 GPO triplets coupled to a NC1(XIX) domain was produced as described [13]. Cell cultures MRC-5, a human lung embryonic fibroblast cell line, was provided by RD-Biotech collection. BZR and MG-63 cells, two human bronchial epithelial and osteosarcoma cell lines, were provided by the American Type Culture Collection (ATCC, Manassas, VA). They were grown in Dulbecco’s modified eagle medium (DMEM) supplemented with 5% fetal bovine serum (FBS) in Nunclon 25 cm2 flasks (Dutscher, Brumath, France) at 37 °C in a humid atmosphere composed of 5% CO2 and 95% air. Sample preparation Kidney tissue samples were obtained from Laboratory of Anatomy of the Faculty of Medicine. Extraction of collagen XIX from tissue samples was performed according to Myers et al. [14]. The anatomical piece was rinsed several times in a solution containing 50 mM Tris-HCl, 4.5 M NaCl, 20 mM EDTA, pH 7.5, 10 mM N-ethylmaleimide, 1 lg/mL aprotinin (w/v). It was then cut into multiple fragments of less than 0.5 cm, and placed in an extraction buffer at 4 °C for 24 h (30 ml, 10/1 v/w) containing 50 mM Tris-HCl, 1 M NaCl, 10 mM EDTA, pH 7.5, and protease inhibitors: 5 mM N-ethylmaleimide, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM benzamidine, 0.1% azide. The tissue was homogenized on ice using an Ultra-Turrax T25 (8  1 min). The tissue suspension was stirred slowly for 24 h at 4 °C. After 24 h, the extract was centrifuged at 32,000 rpm for 30 min. The supernatant was collected and the protein supernatant was then precipitated with ammonium sulfate to 40% saturation, and then centrifuged again at 32,000 rpm for 30 min at 4 °C. The protein pellet thus obtained was resuspended in 20 mL of extraction buffer containing 0.1% Triton X-100 and slowly stirred overnight at 4 °C and then dialyzed overnight at 4 °C against 500 mL of solution containing 25 mM Tris-HCl, 0.4 M NaCl, 2.0 mM EDTA, pH 7.4, Triton X-100 (0.1%), and protease inhibitors: 5 mM N-ethylmaleimide, 0.5 mM PMSF, 5 mM benzamidine, 0.1% azide. A second dialysis was then performed against 500 ml of the same buffer except pH (7.2) and NaCl (0.1 M) concentration. The dialyzate was centrifuged at 20,000 rpm for 15 min at 4 °C. The supernatant was incubated in a batch procedure with Sepharose beads (Sepharose Fast Flow Resin S, Amersham Biosciences), preequilibrated in the final dialysis buffer containing 0.1 M NaCl, overnight at 4 °C with gentle stirring. The suspension was centrifuged at 480g for 5 min at 4 °C. The resin was washed with a buffer containing 25 mM Tris-HCl, 2.0 mM EDTA, pH 7.2, 5 mM N-ethylmaleimide, 0.5 mM PMSF, 5 mM benzamidine, 0.1% azide. Elutions were carried out stepwise at 30-min intervals using increasing concentrations of NaCl (0.1 to 1 M) with centrifugation at 480g, 5 min at 4 °C. Conventional PCR RNA isolation was performed using the Qiagen RNeasy kit (Qiagen, Courtaboeuf, France) according to the manufacturer’s instructions. cDNA was prepared from 1 lg of total RNA by reverse transcription (RT) at 42 °C for 45 min. The primers were

[50 -ctggtcaaaagggagagcaa-30 ] and [50 -ctcctttatgccctttct-30 ] for COL19A1, [50 -ctggagccaagtgctaacatgcc-30 ] and [50 -ccgggtttgagaacaccagtc-30 ] for EEF1A1. The primers were designed using the eprimer 3 software and the specificity of the sequences was checked using the NCBI Blast software. The PCR was performed in an Eppendorf thermocycler and the cycling program (45 cycles) included a 20-s denaturation step at 95 °C and a 30-s annealing step at 60 °C, followed by a 30-s elongation step at 72 °C. The amplified DNA fragments were visualized after agarose gel electrophoresis in the presence of ethidium bromide and image acquisition was performed using the Bio-Capt and Bio-1D software (Vilber Lourmat, Marne la Vallée, France). Quantitative real-time PCR RNA isolation was performed as previously described. SYBR Premix Ex Taq (Ozyme, Saint Quentin en Yvelines) was used for the PCR. The primers used were the same as those above. Mx 3005P (Stratagene) was used for amplification and data collection. A first denaturation step was performed for 10 min at 95 °C, and then the cycling program (45 cycles) included a 5-min denaturation step at 95 °C, a 30-s annealing step at 60 °C, followed by a 15-s elongation step at 72 °C. Fluorescence acquisition was carried out at 72 °C in single mode at the end of the elongation step. After real-time PCR, a melting-curve analysis was performed by continuously measuring fluorescence during heating from 55 to 95 °C at a transition rate of 0.2 °C/s. Product specificity was evaluated by melting-curve analysis and by electrophoresis in 2% agarose gel. Fluorescence was analyzed by the Data Analysis software (Stratagene). Crossing points (Cp or Ct) were established using the second derivative method. The qPCR efficiency was calculated from the slope of the standard curve. Target gene expression levels were normalized to reference gene. The results were calculated using the delta-delta method. Western blot For Western blot analysis, samples, reduced or not by 10 mM dithiothreitol, were subjected to SDS-PAGE (0.1% SDS, 10% polyacrylamide gel) (100 lg total protein per lane), and then transferred onto Immobilon-P membranes (Millipore, St. Quentin en Yvelines, France). Membranes were blocked by incubation with 5% nonfat dry milk, 0.1% Tween 20 in 50 mM Tris-HCl buffer, 150 mM NaCl, pH 7.5, for 2 h at room temperature. They were incubated overnight with a 1/1000 diluted rabbit anti-human NC1(XIX) antibody and then for 1 h with the 1/10000 diluted corresponding peroxidase-conjugated second antibody at room temperature. Immune complexes were visualized using the ECL prime chemiluminescence detection kit (GE Healthcare, Orsay, France). Competitive ELISA procedure For determining ELISA conditions, 96-well microtiter plates were used for enzyme-linked immunosorbent assay. Serial dilutions were performed from a single stock solution to obtain the different dilutions of antibody and peptide concentrations. NC1(XIX) peptide (0 to 20 lg/well) was adsorbed to the 96-well microtiter plate in carbonate buffer (0.2 M sodium carbonate, 0.2 M sodium bicarbonate, pH 9.6) overnight at 4 °C. After washing with TBST (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, Tween 20 0.05% (v/v)), each well was blocked with TBST-BSA 1% (TBST-bovine serum albumin) for 2 h at 37 °C. After washing the plate with TBST, 100 ll of the primary antibody solution (IgG rabbit anti-NC1 (XIX)) at different dilutions (1/100 to 1/800) in TBST-1% BSA was added to each well, and then incubated for 1 h at room

Collagen XIX measurement in biological fluids/J.B. Oudart et al. / Anal. Biochem. 437 (2013) 111–117

temperature. After three washes with TBST, 100 lL of the secondary antibody solution (anti-rabbit IgG coupled to peroxidase) diluted 1/5000 was added to each well, and then incubated for 1 h at room temperature. After three washes, 100 lL per well of TMB (tetra-methylbenzidine), a peroxidase substrate, was added to the wells and incubated for 30 min in the dark. The enzymatic reaction was stopped by adding 50 lL per well of 0.5 M H2SO4. The intensity of the yellow coloration was measured at 450 nm with LB940 spectrophotometer (Berthold Technologies, Yvelines, France). Competitive enzyme-linked immunosorbent assay was performed according to Signorella and Hymer [15]: Step 1 (Coating): NC1(XIX) peptide (200 ng/well) was adsorbed to the 96-well microtiter plate in carbonate buffer (0.2 M sodium carbonate, 0.2 M sodium bicarbonate, pH 9.6) overnight at 4 °C. After washing with TBST (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20 (v/v)), each well was blocked with TBST-BSA 1% (TBST-bovine serum albumin) for 2 h at 37 °C. Step 2 (Competition): 60 lL of each sample or calibrator was incubated with 60 lL of primary antibody (IgG rabbit antiNC1 (XIX) IgG) diluted 1/400e in TBST-1% BSA for 1 h at room temperature. Step 3: 100 lL of each competitive mixture from step 2 was added in wells coated with NC1(XIX) in step 1. The plate was incubated for 1 h at room temperature. Step 4: After three washes with TBST, 100 lL of secondary antibody solution (anti-rabbit IgG coupled to peroxidase) diluted 1/ 5000e was added to each well, and then incubated for 1 h at room temperature. Step 5: After washing with TBST, 100 lL per well of a peroxidase substrate, TMB, was added and incubated in the dark for 30 min. Step 6: The enzymatic reaction was stopped by adding 50 lL per well of 0.5 M H2SO4. The intensity of the yellow coloration was measured at 450 nm with LB940 spectrophotometer (Berthold Technologies, Yvelines, France).

Results Conventional PCR For the development of conventional PCR, we used three cell lines: bronchial epithelial cells (BZR), osteosarcoma cells (MG63), and embryonic lung fibroblasts (MRC-5), usually described to synthesize extracellular matrix components and potentially express collagen XIX alpha 1 chain gene (COL19A1). Using collagen COL19A1 specific primers, several temperatures of primer annealing were tested to optimize the sensitivity and specificity of the technique (56, 58, 60, 62 °C). The more efficient annealing temperature was 60 °C (data not shown). Under these conditions, we obtained for the three tested cell lines a single band of 176 bp for the COL19A1 amplicon, as expected (Fig. 1A). Quantitative real-time PCR The qPCR assays were optimized using DNA obtained from MRC-5 cell cultures. The specific amplification of a 176-bp fragment was performed by qPCR using collagen XIX specific primers. The amplification of different cDNA dilutions (1/5, 1/25, 1/125, 1/ 625, 1/3125) (Fig. 1B) was performed in order to determine the PCR efficiency. The melting-curve analysis (Fig. 1C) showed an amplification product with a (Tm) of 84.5 °C corresponding to the collagen XIX amplicon. A standard curve of collagen XIX was performed (Fig. 1D). The assay was linear and the value of amplification efficiency was 96.1% with a slope of 3.41 and a R2 coefficient of 0.99. Western blot The Western blot assay was performed and optimized for protein extract from kidney, various human fluids (serum, umbilical cord blood, and amniotic fluid), and MRC-5 cell culture medium. A rabbit polyclonal anti-human NC1(XIX) was produced by Covalab. The NC1(XIX) peptide, NPEDCLYPVSHAHQRTGGN, specific to

Φx 174

B

221 bp

176 bp

Fluorescence (dRn)

MG63

BZR

EEF1A1 MRC5

MG63

MRC5

A

BZR

COL19A1

113

Cycles

D

Ct (dRn)

Fluorescence (Rn’(T))

C

Temperature (°C)

Initial Quantity (relative)

Fig.1. (A) Conventional PCR analysis of collagen XIX expression. MRC-5, human embryonic lung fibroblasts; BZR, bronchial epithelial cells; MG-63, osteosarcoma cells. (B) Real-time PCR amplification curves of serial MRC-5 cell DNA dilutions. (C) Derivative melting curve of serial MRC-5 cell DNA dilutions. (D) Real-time PCR standard curve

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MRC-5 culture medium reducing conditions

MRC-5 culture medium non reducing

A

1

2

3

B

4

5

6

7

8

Fig.2. Western blot analysis of MRC-5 culture medium using an anti-collagen XIX antibody under nonreducing (A) (1, 2, 3, 4: replicates) and reducing (B) conditions (5, 6, 7, 8: replicates).

as control (Fig. 3E). Preincubation of the samples with collagenase induced a significant decrease of the signal in Western blot (Fig. 3F). All these results demonstrate the affinity of the primary antibody.

collagen XIX, was used for rabbit immunization. In nonreduced MRC-5 culture medium, a 250-kDa band corresponding to the intact collagen XIX trimeric form was observed (Fig. 2A). Under reducing conditions, a second band appeared at 145 kDa, corresponding to the alpha 1 chain of collagen XIX (Fig. 2B). These results are in agreement with the literature [8]. In kidney tissue extracts, three bands (70, 50, and 45 kDa) corresponding to proteolytic products of collagen XIX in vivo are shown (Fig. 3A). In the various biological fluids used, two bands at 100 and 70 kDa were found, corresponding to proteolytic cleavage products of collagen XIX. To demonstrate the affinity of our primary antibody, we performed preincubation of the antibody solution with increasing concentrations of NC1(XIX) peptide (500 to 2000 lg/mL) for 30 min at room temperature on a rocking platform. This preincubation induced a significant decrease of the signal in Western blot (Fig. 3B–D). Moreover, one major band with a Mr 55 kDa was observed with the trimeric mini-collagen [(GPO)6 - NC1-(XIX)]3 used

Determination of ELISA conditions For competitive ELISA assay, we first determined the optimal primary antibody concentration to use and the optimal amount of peptide to coat onto the plate. This was determined using different concentrations of the primary antibody (1/100, 1/200, 1/400, and 1/800) and different amounts of coated peptide (0 to 20560 ng/well). The calibration curve was performed using serial dilutions of a single stock solution of peptide. The different steps were 0, 20, 40, 80, 160, 320, 640, 1280, 2560, 5120, 10280, and 20560 ng/well (Fig. 4) We selected the conditions to have an absorbance of 1 unit, that are a dilution of 1/400 of the primary antibody

A 70 kDa 50 kDa 45 kDa

B

Without competition

C

D

500 μg NC1(XIX) competition

2000 μg NC1(XIX) competition

100 kDa

70 kDa Amniotic Fluid

Umbilical cord blood

Amniotic Fluid

Umbilical cord blood

Amniotic Fluid

Umbilical cord blood

E collagenase

-

+

-

+

100 kDa 70 kDa 55 kDa

Amniotic Fluid

Mini Collagen XIX

Fig.3. Anti-NC1(XIX) Western blot analysis of 3 different kidney tissue extracts (A). Anti-NC1(XIX) Western blot analysis of amniotic fluid and umbilical cord blood without competition (B), after competition with 500 lg of NC1(XIX) (C) and after competition with 2000 lg of NC1(XIX) (D). Anti-NC1(XIX) Western blot of the samples treated or not with collagenase (E).

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Fig.4. Direct ELISA: Intensity of signal obtained with different primary antibody concentrations and different amounts of peptide coated. }, h, 4, s: antibody dilutions 1/100, 1/200, 1/400, and 1/800, respectively.

and a coating of 200 ng/well of NC1 (XIX) (Fig. 4). This combination was selected because it gave an absorbance of 1 unit using the lowest amount of peptide and the highest dilution of antibody. Competitive ELISA The calibration curve was determined with increasing concentrations of NC1(XIX) peptide from 0 to 40 lg/mL (Fig. 5A). To determine the peptide concentration in the samples, we performed a logarithmic transformation of this curve, as usually done in the

literature (Fig. 5B). The obtained calibration curve was linear (y = 26.856X + 125.07 and R2 = 0.97). The lower quantifiable concentration corresponded to 0.019 lg/mL. To validate the accuracy of our competitive ELISA, we measured several serum samples with known NC1(XIX) concentrations. The measured concentrations were well correlated with the expected concentration (Table 1). The optimized validated ELISA was used to determine NC1(XIX) levels in the sera of 38 different donors. NC1(XIX) concentrations were measured in triplicate or quadruplicate and varied from 0

A

B

Fig.5. ELISA calibration: The calibration curve was determined with increasing concentrations of the NC1(XIX) peptide, from 0 to 10 lg/mL (A). Logarithmic transformation of the calibration curve was linear (y = 26.856X + 125.07 and R2 = 0.97) (B).

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Collagen XIX measurement in biological fluids/J.B. Oudart et al. / Anal. Biochem. 437 (2013) 111–117 Table 1 Measurement by competitive ELISA of several serum samples with known concentrations of NC1(XIX) added. Samples

Sample’s absorbance

Inhibition (A/A0)x100

Expected concentration (lg/mL)

Measured concentration (lg/ml)

% Error

Sample Sample Sample Sample Sample Sample

0.152 0.263 0.387 0.397 0.514 0.637

14.2 24.6 36.3 37.2 48.2 59.7

10 5 2.5 1.25 0.625 0.3125

12.60 5.70 2.18 1.11 0.54 0.32

26 14 13 11.2 14 2.72

1 2 3 4 5 6

NC1 XIX concentration (ng/mL)

The measured concentrations were well correlated with the expected concentrations. The absorbance without competition (A0) was 1.067.

950 900 850 800 750 700 650 600 550 500 450 400 350 300 250 200 150 100 50 0 Sera from donors

Fig.6. ELISA measurement of NC1(XIX) concentration in sera from different donors. Serum samples from 38 patients were assessed for their type XIX collagen content.

to 858 ng/ml, depending on the patient. Fig. 6 shows a simple plot for each patient which corresponds to the average of triplicate or quadruplicate measurements. The error calculation was performed on a set of two independent assays using 38 samples measured in triplicate or quadruplicate. The calculated error was 10.3 ± 4.9%. These preliminary results were only used to validate the development of the assay in serum. We did not expect to highlight physiological or pathological changes in such a shortage of donors. However, a clinical trial is currently underway to search possible variations of expression in various pathological conditions.

the presence of autoantibodies directed against the a3(IV) collagen chain [22]. Measurement of collagen IV in serum by ELISA was also proposed as an early marker of renal diseases, especially of nephropathy, in diabetic patients [23,24]. Type XIX collagen is a minor collagen associated with basement membranes. The development of robust technologies for assessing its expression in various tissues and fluids is required. For that purpose, we developed a real-time PCR assay, Western blot analysis, and competitive enzyme-linked immunosorbent assay. These methodologies will enable evaluation of collagen XIX in various clinical situations (cancer, vascular pathology, pregnancy, or renal diseases). Actually, the techniques that are presented in this paper constitute an integrated set of assays that appreciate collagen XIX metabolism in a series of pathologies in which basement membrane alterations may occur. The combination of qPCR, Western blot, and ELISA assays gives a complete understanding of type XIX collagen expression in tissues and body fluids, whereas individual techniques give only a partial vision. They may be used in various human tissue extracts and biological fluids such as serum, amniotic fluid, cord blood, and many other liquids. Collagen XIX assay could be useful for the diagnosis or prognosis of diseases in which basement membrane metabolism may be altered. Acknowledgments This work was supported by Grants from the Université de Reims-Champagne-Ardenne, the Centre National de la Recherche Scientifique, the Ligue Nationale contre le Cancer, the Region Champagne-Ardenne, and the Fonds Européen de Développement Régional (FEDER). References

Discussion Extracellular matrix protein functions, and especially those of basement membrane constituents, are increasingly identified [1,2]. Extracellular matrix constituents are involved in many pathophysiological situations such as inflammation, cell migration, tumor invasion, angiogenesis, embryonic development, and others. The diagnosis of several diseases is partly based on the measurement of different circulating molecules. Such proteins or their proteolytic fragments can be measured in biological fluids such as blood or urine, and these assays may be used for diagnosis, prognosis, or treatment monitoring [16,17]. For example, serum endostatin (a fragment of collagen XVIII) measurement is used in clinical practice to predict tumor vascularity [18,19]. Endostatin is also currently used in clinical trials as an additional anticancer therapy [20]. Another example is collagen IV, the major constituent of basement membranes. Collagen IV is involved in various diseases, for instance, Alport syndrome, characterized by different COL4A5 gene mutations [21], or in Goodpasture’s syndrome, characterized by

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