Medizinische Hochschule Hannover
Institut für Klinische Chemie
Cellular and molecular characterization of the effects of different anticoagulants on the regulation of matrix metalloproteinase 9
INAUGURALDISSERTATION zur
Erlangung des Grades einer Doktorin der Humanbiologie
‐ Doctor rerum biologicarum humanarum – (Dr. rer. biol. hum.)
vorgelegt von
Rozan Romel Attili/Abedalkhader aus Jeddah/Saudi‐Arabien
Hannover 2016
II
Angenommen vom Senat der Medizinischen Hochschule Hannover am 01.08.2016
Gedruckt mit Genehmigung der Medizinischen Hochschule Hannover
Präsident: Prof. Dr. med Christopher Baum Betreuer: Prof. Dr. rer. nat. Ralf Lichtinghagen Kobetreuer: Prof. Dr. med. Karin Weißenborn Referent: Prof. Dr. rer. nat. Ralf Lichtinghagen Korrferent: Prof. Dr. med. Karin Weißenborn Korrferent: Prof. Dr. med. Andreas Tiede
Tag der mündlichen Prüfung vor der Prüfung vor der Prüfungskommission: 01.08.2016
Prof. Dr. rer. Boil. hum. Ronald Jacobs Prof. Dr. rer. nat. Ralf Lichtinghagen Prof. Dr. med. Karin Weißenborn Prof. Dr. med. Andreas Tiede
Wissenschaftliche Betreung:
Prof. Dr. rer. nat. Ralf Lichtinghagen Wissenschaftliche Zweitbetreuung:
Prof. Dr. med. Karin Weißenborn
1. Gutachter: Prof. Dr. rer. nat. Ralf Lichtinghagen, Institut für Klinische Chemie, Medizinische Hochschule Hannover
2. Gutachterin: Prof. Dr. med. Karin Weißenborn, Klinik für Neurologie, Medizinische Hochschule Hannover
3.Gutachter: Prof. Dr. med. Andreas Tiede, Klinik für Hämatologie, Hämostaseologie, Onkologie und Stammzelltransplantation
Tag der mündlichen Prüfung: 01.08.2016
III
Index
Abbreviations ……….………XI
List of Figures………..XV List of Tables……….XVII
1. Introduction……….1
1.1Matrix Metalloproteinases (MMPs)‐History………1
1.2 Biochemistry and structural complexity of MMPs……….3
1.3 The phenotype of KO mice……….6
1.4 Regulation of MMPs……….7
1.4.1 Factors inducing and inhibiting MMPs………...8
1.4.2 Transcription factors regulating MMP‐9 expression………9
1.4.3 Regulation by structure………..11
1.4.4 Regulation by inhibitory proteins……….12
1.4.5 Regulation by further agents……….………….13
1.5 The biological roles of the MMPs……….13
1.6 Matrix metalloproteinase‐9 ………15
1.7Structure and regulation of MMP‐9………15
1.7.1 Structure of MMP‐9………..15
IV
1.7.2 Regulation of MMP‐9………16
1.7.2.1 Cytokines and growth factors……….16
1.7.2.2 Signal transduction……….17
1.7.2.3 Transcription factors……….19
1.8 Role of MMP ‐9 in malignancies and cardiovascular diseases……….21
1.9 MMP‐9 as a biomarker……….23
1.10 Aims of the study………..25
2. Materials & Methods………26
2.1 Materials………26
2.1.1 Chemical reagents………..26
2.1.2 Enzymes……….26
2.1.3 DNA size markers………27
2.1.4 Laboratory equipment……….27
2.1.5 Buffers……….28
2.1.6 Commercial kits………29
2.1.7 Software………29
2.2 Methods……….30
2.2.1 Blood specimen collection and storage………30
V
2.2.2 Cell culture………31
2.2.2.1 Cell lines……….31
2.2.2.1.1 Individual culture………31
2.2.2.1.2 Stimulation with T‐cell‐derived supernatant………..32
2.2.2.1.3 Stimulation of individual monocyte cultures with the supernatant of T‐cells stimulated with derived human plasma derived from heparin monovettes ………32
2.2.2.1.4 Double co‐culture experiments...……….32
2.2.2.1.5 Triple co‐culture experiments….………..33
2.3 Anticoagulants……….33
2.3.1 HMWH………33
2.3.2 LMWH……….33
2.3.3 Citrate………34
2.3.4 EDTA………34
2.4 RNA isolation and cDNA synthesis………34
2.4.1RNA isolation and purification………..……….34
2.4.2RNA quantification using Nano‐Drop ND ‐1000……….35
2.4.3 cDNA synthesis………..35
2.4.4 Agarose gel electrophoresis……….35
2.5 Detection of mRNA expression ……….36
VI
2.5.1Gradient polymerase chain reaction……….36
2.5.2 Quantitative reverse transcription PCR (qRT‐PCR)………..37
2.6 Detection of protein expression………37
2.6.1 ELISA……….37
2.7 Identification of secreted soluble mediators………38
2.7.1 Proteome profiler human XL cytokine array………38
2.8 Stimulation of monocytes with identified soluble mediators………..39
2.9Cellular functions of activated monocytes……….40
2.9.1 Proliferation……….40
2.9.2 Phagocytosis………40
2.9.3 Apoptosis………..41
2.10Statistical analysis………42
3 Results………43
3.1 Direct stimulation of different cell types with anticoagulants……….43
3.2 Significant Induction of MMP‐9 expression by HMWH in a co‐culture including THP‐1, Jurkat, and HT cells………..47
3.3 Significant induction of MMP‐9 expression by HMWH in the co‐culture of THP‐1 and Jurkat cells………49
VII
3.4 Significant induction of MMP‐9 expression in THP‐1 cells in response to culture supernatant
derived from HMWH‐treated Jurkat cells………52
3.5 Significant induction of MMP‐9 expression in THP‐1 cells by incubation with supernatant from Jurkat cells stimulated with human plasma derived from HMWH‐containing monovettes………..53
3.6 High molecular weight heparin versus Low molecular weight heparin……….………54
3.7 Identification of T‐cell derived soluble mediators which activate MMP‐9 expression in THP‐1 cells……….57
3.8 HMWH versus LMWH: identified soluble mediators………58
3.9 Regulation of MMP‐9 and IL‐8 expression in THP‐1 by the identified soluble mediators………60
3.10 Identification of Serpin E1 and/or MIF as alternative supporting factor for MMP‐9 induction ………63
3.11Cellular functions of activated THP‐1……….68
3.11.1 Proliferation……….68
3.11.2 Phagocytosis……….69
3.11.3 Apoptosis………70
4 Discussion………..……….73
4.1 Topic overview………..73
VIII
4.2 Direct stimulation with anticoagulants has no influence on MMP‐9 expression of monocytes,
T‐cells, and B‐cells………..75
4.3 Significant Induction of MMP‐9 expression by heparin in a co‐culture including monocytes, T‐cells, and B‐cells………76
4.4 Significant induction of MMP‐9 expression by heparin in the co‐culture of monocytes and T‐ cells……….78
4.5 Significant induction of MMP‐9 expression in monocytes in response to culture supernatant derived from heparin‐treated T‐cells or human heparin plasma‐treated T‐cells………79
4.6HMWH versus LMWH………81
4.7 Identification of T‐cell derived soluble mediators which activate MMP‐9 expression in monocytes……….82
4.7.1 Stimulation with individual soluble mediators has no effect on MMP‐9 expression by monocytes……….85
4.7.2 A combination of T‐cell‐derived IL‐16, sICAM‐1, and monocyte‐derived IL‐8 is able to induce MMP‐9 expression in monocytes……….87
4.8Induction of IL‐8 expression in monocytes by T‐cell‐derived mediators………..89
4.9 Cellular functions of activated monocytes……….91
4.9.1 Proliferation……….91
4.9.2 Phagocytosis………92
4.9.3 Apoptosis………..93
IX
4.10 Conclusion……….……….94
4.11 Outlook ... 95
5 Summary ... 98
6 Deutsche Zusammenfassung……….………101
7 References……….………104 8 Acknowledgments……….……….XVIII 9 Curriculum Vitae……….………...XIX
10 Erklärung………..…….XXIII
X
Abbreviations
°C Grade Celsius
μ Micro
aa Amino acid
AP‐1 Activator proteins ‐1 ATP Adenosine tri‐phosphate BSA Bovine serum albumin C5a Complement factor 5a
Ca2+ Calcium ion
C/EBP CCAAT/enhancer‐binding protein
ConA Concanavalin A
Cys Cysteine array
Da Dalton
DAPI 4′, 6‐Diamidin‐2‐phenylindol ECM Extra cellular matrix
EDTA Ethylene diamine tetra acetic acid EGF Epidermal growth factor
XI
ELISA Enzyme linked immunosorbent assay
EMMPRIN Extracellular matrix metalloprotease inducer
ER Estrogen receptor
ERK Extra cellular signal regulated kinases et al. And others
F Fibronectin repeats
FGF Fibroblast growth factor
Fr Furin
GAPDH Glycerin aldehyde‐3‐phosphate‐dehydrogenase CD Cluster of differentiation
GPI Glycosyl phosphatidyl inositol
h Hour
HMWH High molecular weight heparin
Hpx Hempoxin
IgG‐like Immunoglobulin‐like
IL Interleukin
INF Interferon
Kb Kilo base
XII
KDa Kilo Dalton
KO Knock‐out
KRE Keratinocyte differentiation‐factor responsive element
l Liter
LMWH Low molecular weight heparin LPS Lipopolysaccharide
M Meter
m Milli
M Molar
MAP Mitogen‐activated protein MI Myocardial infraction
MIF Macrophage inhibiting factor MMP Matrix metalloproteinase
MT‐MMP Membrane type matrix metalloproteinase NF‐ B Nuclear factor of B site
PAIs Serine proteinase inhibitors PBS Phosphate buffered saline
PEA3 Polyomavirus enhancer‐A‐binding‐protein‐3
XIII
PCOLCE Pro‐collagen C‐terminal proteinase enhancer protein PDGF Platelet‐derived growth factor
RASI‐1 Rheumatoid arthritis synovium inflamed‐1 RCE Retinoblastoma control element
RECK Reversion including cysteine rich protein with Kazai motif RPMI Roswell Park Memorial Institute
TAE Tris acetate‐EDTA TATA TATA‐box
TIE Transforming growth factor b inhibitory element TIMP Tissue inhibitor matrix metalloproteinases TNF Tumor necrosis factor
VEGF Vascular endothelial growth factor Zn2+ Zinc ions
XIV
List of Figures
Fig. 1.1: Domain structure classifications of Mammalian MMPs………..6
Fig 1.2: Levels of regulation of MMP expression and activity………..9
Fig. 1.3: Regulatory elements in the promoter regions of human MMP gene……….11
Fig. 1.4: Schematic structure of MMP‐9………...16
Fig 1.5: Signaling pathways involved in MMP gene transcription and potential strategies for therapeutic intervention……….18
Fig 1.6: Regulatory elements in the promoter regions of MMP‐9……….19
Fig 1.7: Strategies for blocking pro‐MMP activation………21
Fig. 3.1: Direct anticoagulant stimulation has no stimulatory effect on MMP‐9 expression in THP‐1 cells……….44
Fig. 3.2: Direct anticoagulant stimulation has no stimulatory effect on MMP‐9 expression in Jurkat cells……….45
Fig. 3.3: Direct anticoagulant stimulation has no stimulatory effect on MMP‐9 expression in HT cells……….46
Fig. 3.4: Effect of anticoagulant‐stimulation on the MMP‐9 mRNA expression in a co‐culture of THP‐1, Jurkat, and HT cells……….48
Fig. 3.5: Effect of anticoagulant‐stimulation on the MMP‐9 mRNA expression in a co‐culture of THP‐1, Jurkat, and HT cells stimulated with HMWH‐, EDTA‐, or citrate‐treated supernatant of Jurkat or HT cells………48
XV
Fig. 3.6: Effect of EDTA and citrate stimulation on the MMP‐9 mRNA expression in a co‐culture of THP‐1 and Jurkat or THP‐1 and HT cells……….50 Fig. 3.7: Effect of HMWH stimulation on the MMP‐9 mRNA expression in a co‐culture of THP‐1 and HT cells or Jurkat and HT‐cells treated with HMWH……….51 Fig. 3.8: Effect of HMWH stimulation on the MMP‐9 mRNA expression in a co‐culture of THP‐1 and Jurkat cells………51 Fig. 3.9: Induction of MMP‐9 expression in THP‐1 cells by incubation with supernatant from HMWH‐stimulated Jurkat cells………53 Fig. 3.10: Induction of MMP‐9 expression in THP‐1 cells by incubation with supernatant from Jurkat cells stimulated with human heparin‐plasma………54 Fig. 3.11: Direct stimulation of THP‐1 with HMWH or LMWH (Clexane or Fragmin) has no MMP‐
9‐inducing effect………..55 Fig. 3.12: LMWH (Clexane or Fragmin) has no significant MMP‐9–inducing effect in a mixture of THP‐1 and Jurkat cells………56 Fig. 3.13: Identification of cytokines/chemokines expressed by THP‐1, Jurkat, and HT cells in
response to HMWH……….58
Fig. 3.14: Identification of soluble mediators expressed by HMWH‐and LMWH‐treated THP‐1 or THP‐1 and Jurkat cells………60 Fig. 3.15: Influence of individual soluble mediators on MMP‐9 expression in THP‐1 cells………..61
XVI
Fig. 3.16: Influence of combinations of soluble mediators on MMP‐9 expression in THP‐1
cells……….62
Fig. 3.17: Identification of Serpin E1 and/or MIF as alternative supporting factors for MMP‐9 induction……….64
Fig. 3.18: Influence of T‐cell‐derived soluble mediators on IL‐8 production by THP‐1 cells………65
Fig. 3.19: Influence of combinations of T‐cell derived soluble mediators on THP‐1 cells………….66
Fig. 3.20: THP‐1 proliferation is induced by HMWH‐treated Jurkat supernatant………69
Fig. 3.21: THP‐1 phagocytosis is enhanced by HMWH‐treated Jurkat supernatant………..70
Fig. 3.22: Apoptosis is enhanced in THP‐1 by HMWH‐treated Jurkat supernatant………71
Fig 4.1: Secretion of soluble mediators by T‐cells in response to HMWH………80
Fig 4.2: Induction of MMP‐9 expression by monocytes in response to T‐cell‐derived IL‐16 and sICAM‐1 is supported by autocrine production of monocytic IL‐8………91
List of Tables Tab. 1: MMPs in Human……….1
Tab.2: PCR reaction pipetting procedure……….36
Tab. 3: Proteome profiler human XL cytokine array supplemented membrane……….39
XVII
1 Introduction
1.1 Matrix Metalloproteinases ‐ History
Matrix metalloproteinases (MMPs) are a family of enzymes which have the potential to degrade the extracellular matrix (ECM) and the basement membrane components (1). Since the discovery of tadpole collagenase that could degrade fibrillar collagen during metamorphosis in 1962 by Gross and Lapiere, MMPs have emerged as a significant proteinase group with recognized effects on the cardiovascular system (2). In 1986, collagenase‐1 was identified and sequenced from human skin as a first enzyme of the human MMP family, which was able to cut the triple helix at a point one‐quarter of the distance from the C‐terminal end (3). To date, 24 different vertebrate MMPs have been identified of which 23 are found in humans (Table 1). These proteinases play a central role in many biological processes.
Tab. 1: MMPs in humans
Enzyme Descriptive name Location Description
MMP‐1 Interstitial collagenase Secreted Substrates include Col I, II, III, VII, VIII, X, gelatin
MMP‐2 Gelatinase‐A, Secreted Substrates include Gelatin, Col I, II, III, IV, Vii, X 72 kDa gelatinase
MMP‐3 Stromelysin 1 Secreted Substrates include Col II, IV, IX, X, XI, gelatin
MMP‐7 Matrilysin, PUMP 1 Secreted membrane associated through binding to cholesterol sulfate in cell membranes, substrates include: fibronectin, laminin, Col IV, gelatin
MMP‐8 Neutrophil collagenase Secreted Substrates include Col I, II, III, VII, VIII, X, aggrecan, gelatin
MMP‐9 Gelatinase‐B, Secreted Substrates include Gelatin, Col IV, V 92 kDa gelatinase
1
MMP‐10 Stromelysin 2 Secreted Substrates include Col IV, laminin, fibronectin, elastin
MMP‐11 Stromelysin 3 Secreted MMP‐11 shows more similarity to the MT‐MMPs, is convertase‐
activatable and is secreted therefore usually associated to convertase‐
activatable MMPs. Substrates include Col IV, fibronectin, laminin, aggrecan
MMP‐12 Macrophage Secreted Substrates include elastin, fibronectin, Col IV Metalloelastase
MMP‐13 Collagenase 3 Secreted Substrates include Col I, II, III, IV, IX, X, XIV, gelatin
MMP‐14 MT1‐MMP Membrane‐associated type‐I transmembrane MMP; substrates include gelatin, fibronectin, laminin
MMP‐15 MT2‐MMP Membrane‐associated type‐I transmembrane MMP; substrates include gelatin, fibronectin, laminin
MMP‐16 MT3‐MMP Membrane‐associated type‐I transmembrane MMP; substrates include gelatin, fibronectin, laminin
MMP‐17 MT4‐MMP Membrane‐associated glycosyl phosphatidylinositol‐attached; substrates include fibrinogen, fibrin
MMP‐18 Collagenase 4, ‐ No known human orthologue
xenopuscollagenase
MMP‐19 RASI‐1 ‐
(stromelysin‐4)
MMP‐20 Enamelysin Secreted
MMP‐23 CA‐MMP Secreted type‐II transmembrane cysteine array MMP‐24 MT5‐MMP Membrane‐associated type‐I transmembrane MMP
MMP‐25 MT6‐MMP Membrane‐associated glycosyl phosphatidylinositol‐attached
MMP‐26 Matrilysin‐2, ‐ Endometase
MMP‐27 MMP‐22, C‐MMP ‐
MMP‐28 Epilysin Secreted Discovered in 2001 and given its name due to have been discovered in human keratinocytes. Unlike other MMPs this enzyme is constitutively expressed in many tissues (highly expressed in testis and at lower levels in lung, heart, brain, colon, intestine, placenta, salivary glands, uterus,skin).
A threonine replaces proline in its cysteine switch.
2
1.2 Biochemistry and structural complexity of matrix metalloproteinases
MMPs belong to a larger family of proteases known as the metzincin superfamily of zinc‐ dependent endopeptidases. Collectively, MMPs are capable of degrading all kinds of ECM proteins and (with the exception of MMP‐3) are synthesized as inactive pro‐enzymes (zymogens).
MMPs are classified as the matrixin subfamily of zinc metalloprotease. A typical MMP consists of a pro‐peptide of about 80 aa (also including a signal peptide), a catalytic metalloproteinase domain of about 170 aa, a hinge region with variable length, and a hemopexin (Hpx) domain of about 200 aa. Exceptions to this are MMP‐7,MMP‐23, and MMP‐26 which lack the hinge region and the Hpx domain. MMP‐23 has a unique cysteine‐rich domain and an immunoglobulin‐like domain after the metalloproteinase domain. The MMPs are initially synthesized as inactive zymogens and the cleavage of the pro‐peptide is responsible for the active form of the enzyme.
The catalytic domain is an oblate sphere measuring 35 x 30 x 30 Å consisting of about 170 aa. The active site is a 20 Å groove that runs across the catalytic domain. One Zn2+ ion, which is bound by three histidine residues, is found in the conserved sequence HExxHxxGxxH. Hence, this sequence is a zinc‐binding motif. MMP‐2 possesses Fibronectin type II modules inserted immediately before the zinc‐binding motif in the catalytic domain. The hinge region connects the C‐terminal domain with the catalytic domain of a variable length of 17‐63 aa.
The Hpx‐like C‐terminal domain, which shows sequence homology to the serum protein hemopexin, is characterized by a four‐bladed β‐propeller in which the structure provides a large flat surface involved in protein‐protein interactions. This domain determines substrate
3
specificity and is the site for interaction with tissue inhibitors matrix proteinases (TIMPs), but is absent in MMP‐7, MMP‐23, and MMP‐26.
In addition to the C‐terminal domain and the Hpx‐like C‐terminal domain, membrane bound membrane type MMPs (MT‐MMPs) have a transmembrane region. This domain consists of 30 hydrophobic aa residues. MT‐MMPs are not secreted but inserted into the plasma membrane by a transmembrane segment or a C‐terminal Glycosylphosphatidylinositol (GPI) anchor at their C‐
terminal end (4).
The classification of MMPs is based on their substrate specificity in which they can be divided into collagenases, gelatinases, stromelysins, membrane‐bound MMPs, MMP Matrilysine, and unclassifiable subgroups (5).
MMPs share some common characteristics, for example the usualsynthesis as a pro‐enzyme, the activation by proteinases including MMP themselves as an organic mercury compounds, and inhibition by TIMPs, α2–macroglobulin, 110‐phenanthroline, and ovostatin. Another common feature is the usage of a calcium ion for activity and stability, and acting of zinc as an intrinsic catalytic ion.
Collagenases (MMP‐1, MMP‐8, MMP‐13, and MMP‐18) are enzymes dissolving the Gly bonds in the repeating Gly collagen sequence in collagen (6). The key feature of these enzymes is their ability to cleave interstitial collagens I, II, and III. Collagenases can also digest a number of other ECM and non‐ECM molecules. Gelatinases, i.e., gelatinase A (MMP‐2) and gelatinase B (MMP‐ 9), digest gelatins, the denatured collagens. These enzymes have three repeats of a type II fibronectin domain inserted in the catalytic domain, which bind to gelatin, collagens, and
4
laminin (7). MMP‐2, but not MMP‐9, also digests type I, II, and III collagens (8). Stromelysins, i.e., Stromelysin 1 (MMP‐3) and stromelysin 2 (MMP‐10), have similar substrate specificities, but MMP‐3 has a proteolytic efficiency generally higher than that of MMP‐10. MMP‐3 activates a number of pro‐MMPs, e.g. its action on partially processed pro‐MMP‐1 is critical for the generation of fully active MMP‐1 (9). In addition, they are considered as activators of gelatinases, but also take part in the degradation of laminin, fibronectin, and proteoglycans (10)
MMP‐7 processes secreted molecules such as pro‐defences in and pro–tumour necrosis factor (TNF) and is able to cleave a variety of cell surface proteins like E‐cadherin, and Fas‐ligand.
MMP‐2 has the ability to digest a number of ECM components. However, other unclassified MMPs can be categorized under any assigned groups (Fig. 1.1).
5
created in 1989. KO mice are important due to their biological closeness to humans, which allows the analysis of a specific inactivated gene in the mouse and the identification of any difference from the normal behavior or physiology of the respective mice. Moreover, there are several thousand different strains of KO mice (usually named after the gene which has been inactivated) which are widely used for studying various diseases such as cancer, cardiovascular diseases, arthritis, and Parkinson. Additional evidence which supports adetrimental role of MMP‐9 in inflammatory CNS damage has been obtained in animal models. As an example for MMP deficiency, MMP‐9 KO (in the genetic background of C57BL/6mice) has been used as an inflammatory phenotype to study respiratory disease (12). Other MMP‐9 KO in the genetic background of CD1 mice caused a reduction in ischemic lesion volume after permanent focal ischemia (12). More models showed adecreased atherosclerotic burden and impaired macrophage infiltration in the cardiovascular system (13). However, the models also exhibit some limitations, since KO mouse strains can differ and thus, the phenotypes may be divergent due to the variation of genetic backgrounds.
1.4 Regulation of MMPs
Generally, it is quite reasonable that MMPs have to be tightly regulated to avoid unnecessary remodelling and damage to the tissue. Thus, the regulation of the expression of each individual MMP is of major importance. As shown in Fig. 1.2, the proteolytic activity of MMPs is mainly regulated at three levels involving transcription, pro‐enzyme activation, and inhibition by endogenous inhibitors such as TIMPs. The control at the level of transcription is the major means of MMP regulation (14).
7
TIMPs can inhibit most MMPs and as such, one of their roles is to limit proteolysis during ECM remodeling. However, it is now known that this role is not simple. For instance, there are studies showing TIMP‐4 as a potential anti‐angiogenic agent as it is able to inhibit tumor growth, angiogenesis, and metastasis in a xenograft model of mammary tumor genesis in mice (15).
1.4.1 Factors inducing and inhibiting MMPs
The expression of MMPs is positively regulated (stimulated) by various factors including cytokines (e.g., IL‐1, TNF). For instance,TNF stimulates the secretion of active MMP‐2 in a human organ culture model representing full‐thickness human skin and may also affect matrix remodeling during wound healing and other physiological and pathological processes (16).
Moreover,TNF (in combination with IL‐1‐α or IL‐1‐β) leads to a highly synergistic MMP‐3 increase (17). Other inducing factors are chemokines and growth factors including epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), platelet‐derived growth factor (PDGF), and the extracellular matrix metalloprotease inducer (EMMPRIN) (18) as well as physical agents such as ultraviolet‐B irradiation. On the other hand, expression of MMPs is negatively regulated by suppressive factors such as IL‐4, IL‐10, IL‐13, and TGF‐β (19).
This suppression was revealed in IL‐4 which suppresses MMP‐9 expression in human monocytes stimulated with (20). TGF‐β also reduces MMP‐9 expression induced by TNF in MonoMac‐6 (MM‐6) monocytic cells (21). In addition, IL‐10 inhibits MMP‐9 induction by ConA in human monocytes (22).
8
several TF. As shown in MMP‐9 studies both ETS and AP‐1 sites were required for ras‐induced up‐regulation of MMP‐9 promoter activity (23). Moreover, synergistic up‐regulation of MMP‐9 is mediated through interactions of AP‐1 and NF‐κB (24), as shown by specific mutations in the AP‐1 and NF‐κB bindingsites of the MMP‐9 promoter (24). Equivalent results have been obtained in the promoters of MMP‐1 and MMP‐3 (25). The effect of estrogen receptor (ER)‐ coupled c‐fos on MMP‐9 activity was mediated by the proximal AP‐1 site of the promoter. Furthermore, suppression of AP‐1 is responsible for CADPE‐induced inhibition of MMP‐9 induction and cell invasion (26).
A recent study has shown that the inhibition of MMP‐9 expression by H.sabdariffa leaf extract may act through the suppression of the Akt/NF‐kB signaling pathway, which in turn led to the reduced invasiveness of the cancer cells (27). SP‐1 also binds to the MMP‐9 promoter to induce transcription, and the inhibition of SP‐1 leads to decreased MMP‐9 expression (28). Moreover, mutation of either SAF‐1 or AP‐1 binding sites greatly affects induction of the MMP‐9 promoter and reduces the ability of SAF‐1 and AP‐1 to activate transcription (29). In the promoters of other MMP family members, further regulating binding sites have been reported, e.g., for MMP‐2, ‐13,
‐14, and ‐19, polyoma virus enhancer‐A‐binding‐protein‐3 (PEA3) sites are present and in MMP‐1, ‐3, ‐9, 11, 13, and 19, TATA boxes are present in addition to CEBP which is present in MMP‐1, ‐11, and ‐14 (Fig. 1.3).
10
are removed. This leads to a cascade of reactions resulting in conformational changes and the activation of the enzyme (31). This cleavage of cysteine’s through initiation of the cascade is called “cysteine–switch” (32). Most of the MMPs are activated either extracellular by other active MMPs or serine proteinases. For example, MMP‐3 activates pro‐MMP‐1, ‐7, ‐8, ‐9, and ‐ 13, whereas MMP‐2 activates pro‐MMP‐1, ‐9, and ‐13, and MMP‐7 can activate pro‐MMP‐8 and
‐9. In addition, the activation of MMP‐2 is regulated differently and occurs at the cell surface through a unique pathway, which involves MMP‐14 (MT1‐MMP) and TIMP‐2. In this process, pro‐MMP‐2 forms tight complex with TIMP‐2, which is essential for cell activation by MMP‐14 (33). MMP‐11, ‐14, and ‐28, however, can be activated intra‐cellular by furan‐like serine proteinases (34).
1.4.4 Regulation by inhibiting proteins
A third form of MMP regulation is mediated by endogenous specific inhibitors such as TIMPs or α2‐macroglobulin (35). The latter is an abundant plasma protein representing the major inhibitor of MMPs in tissue fluids (36). α2‐macroglobulin plays an important role in the irreversible clearance of MMPs. Inhibitors like TIMPs, however, are more specific and inhibit MMPs in a reversible manner (5). The first TIMP was described in 1975 as a protein in culture medium of human fibroblasts and in human serum, which was able to inhibit collagenase activity (37).
Meanwhile, four different types of TIMPs have been identified (TIMP‐1, ‐2, ‐3, and
‐4).
The interaction between latent MMP‐9 and TIMP‐1 is mediated via the C‐terminal domains of both proteins, whereas the N‐terminal inhibitory domain of TIMP‐1 remains ready to inhibit active MMPs (38, 39). Additionally, latent MMP‐9 is bound to TIMP‐1 before secretion (40).
12
Mainly TIMP‐2 is a bi‐functional protein that acts as an inhibitor of MMPs and also as an activator of pro‐MMPs (41). TIMP‐3, which is tightly bound to the ECM, is involved in cell proliferation, apoptosis, and angiogenesis (42, 43). TIMP‐3 is binds to both pro‐MMP‐2 and pro‐ MMP‐9 (43, 44). In heart, MMP‐9 and TIMP‐4 are predominantly involved in cardiac remodeling by which TIMP‐4 mediates the effect of MMP‐9, moreover, the inhibition of MMP‐9 by either TIMP‐4 or PAR‐1 antagonist or by knocking down the gene itself improved cardiac function (45).
1.4.4 Regulation by further agents
MMPs can be also inhibited by chelating agents (e.g. EDTA), inhibiting antibodies, specific synthetic drugs (e.g. Marimastat), well known drugs with novel MMP inhibitory properties (like bisphosphonates or doxycycline), and chemically modified tetracycline as well as small synthetic peptides (46, 47). The group ofnon‐selective synthetic inhibitors includes the terminal fragment of the pro‐collagen C‐terminal proteinase enhancer protein (PCOLCE) (48) and a GPI‐ anchored glycoprotein called RECK (reversion including cysteine rich protein with Kazai motifs), which is the only known membrane‐bound MMP activation inhibitor (49).
1.5 The biological roles of MMPs
MMPs are important regulators of many biological and pathological processes due to their capability to degrade ECM components. Since ECM remodelling is a critical step in processes such as tissue growth and morphogenesis, MMPs are thought to play important roles during embryonic development, angiogenesis, apoptosis, and morphogenesis of specific tissues (e.g., lung, kidney, and breast) (50).
13
Especially for cell proliferation and angiogenesis, ECM remodelling appears to be important, since for both processes the breakdown of ECM seems to be a prerequisite.
MMPs are also considered to be involved in many pathological conditions such as cancer, cardiovascular diseases, arthritis, nephritis, neurological disease, breakdown of the blood brain barrier, periodontal disease, skin ulceration, gastric ulcer, corneal ulceration, liver fibrosis, emphysema, and fibrotic lung disease (51). For example, in some cardiovascular disease studies, the contribution of MMP‐9 and ‐14 was revealed using MMP‐9‐ and ‐14‐deficient mice which both showed impaired angiogenesis during development (52). MMPs are also key‐players in the precise regulation of many bioactive molecules, mainly by their proteolytic and protein processing activity (11). In this context, it could be shown that MMPs cangenerate growth‐
promoting signals, asdemonstrated by the decreased cell proliferation rates of tumor cells injected into MMP‐9 deficient mice when compared to wild‐type mice (53, 54). Moreover, the proliferation of malignant cells as detected by immunohistochemistry for proliferating cell nuclear antigen is restored by bone‐marrow transplantation with MMP‐9‐positive cells to MMP‐9‐deficient mice (54). Furthermore, MMPs also support metastasis as shown by the increased invasion of certain cell lines through collagen‐containing matrices, when MMP‐2, ‐3, ‐ 13 and –14 were overexpressed (55), (56) . In addition to metastasis, MMPs are involved in the development and progression of arthritis. For instance, MMP‐13 was characterized by an increased expression in ankle joints of WT mice during K/BxN serum‐induced arthritis. Although both K/BxN serum‐treated WT and MMP‐13 KO mice developed progressive arthritis with a similar onset, MMP‐13 KO mice showed significantly reduced disease over the whole arthritic
14
period. These studies suggest that MMPs maybe applied as a potential therapeutic target in certain diseases (57, 58).
1.6 Matrix metalloproteinase‐9
MMP‐9, also known as 92 kDa type IV collagenaseor gelatinase B, was first purified from human macrophages in 1974 (59).The main ability of MMP‐9 is the degradation of gelatin, elastin, collagens IV, V, VII, X, XI, XIV, and fibronectin (60). MMP‐9 is mainly produced by alveolar macrophages, polymorph nuclear leukocytes, osteoclasts, keratinocytes, and invading trophoblasts (61). During human embryonic development, MMP‐9 contributes to the regulation of normal physiological processes, e.g., the morphogenesis of specific tissues such as breast, kidney, and lung (62, 63). MMP‐9 is constitutively expressed in brain and bones, whereas during wound healing, the expression of MMP‐9 has been observed in macrophages, migrating leukocytes, and keratinocytes (64). MMP‐9 has also been described to assist in monocyte migration (65).
1.7 Structure and regulation of MMP‐9
1.7.1 Structure of MMP‐9
In most parts, MMP‐9 possesses the same basic structure like the other MMPs (66). However, several features distinguish MMP‐9 from other MMP family members since it has a gelatin‐
binding domain between the pro‐domain and the catalytic active site. Like other proteolytic enzymes, MMP‐9 is first synthesized as inactive enzyme (i.e., the zymogen) (4).
The included signal pre‐peptide on its N‐terminus is cleaved upon arrival to the endoplasmic reticulum (ER) (67). The remaining pro‐peptide domain contains a short stretch of 10 amino
15
acids which, once attached, maintains enzyme latency by folding over the catalytic domain and interacting with the zinc ion that is responsible for MMP‐9’s enzymatic activity (68). In addition, three repeating fibronectin type‐II sequences inserted in the catalytic site are necessary for the selective binding and cleavage of collagen and elastin by MMP‐9 (69). The hpx‐domain is involved in TIMP‐1 and TIMP‐3 binding and some proteolytic activities (4) (Fig. 1.4).
Fig. 1.4: Schematic structure of MMP‐9 (according to Yabluchanskiy et.al. 2013 (29), modified. The catalytic site contains three essential zinc ion binding sites. At the zymogen stage, a cysteine residue within the pro‐domain interacts with zinc to prevent substrate binding. The haemopexin domain mediates interaction with enzyme substrates. Specific to the gelatinases is the fibronectin‐like domain, which further facilitates substrate binding. MMP, matrix metalloproteinase.
1.7.2 Regulation of MMP‐9
1.7.2.1 Cytokines and Growth Factors
The MMP‐9 mRNA expression is regulated by several cytokines and growth factors (Fig. 1.5).
Among the cytokines capable of regulating MMP‐9 expression, a major important role is assigned to TNF which triggers the production of MMP‐9 through the protein kinase C‐ (PKC‐)
16
dependent signal transduction pathway (70). Additional results indicate that MMP‐9 synthesis and secretion were significantly induced after exposure to the cytokines TNF or IL‐1α, while MMP‐2 levels remained unchanged (71).
MMP‐9 may also be induced via induction by the vascular endothelial growth factor receptor (VEGF‐R1), as shown in lung‐specific metastasis (72). In addition, endothelial growth factor receptor (EGFR) activation also results in the induction of MMP‐9 (73‐75).
In contrast, MMP‐9 suppression is revealed through IL‐4 (20), TGF‐β (21), and IL‐10 (22) (see also chapter 1.1.4.1).
1.7.2.2 Signal Transduction
Mitogen‐activated protein kinases (MAPKs) including extracellular signal‐regulated kinase (ERK), c‐Jun N‐terminal kinase (JNKs), and p38 are part of critical signalling cascades converting upstream signals into biological responses such as cell proliferation, invasion, and transformation (76) (Fig. 1.5). MAPKs regulate MMP‐9 gene expression through transcription factors, e.g., c‐Jun is activated by p38 or JNK. The activated c‐Jun associates with its binding site(s) in the MMP‐9 promoter and subsequently recruits p38 as a cofactor to the promoter to induce its expression.
However, during metastasis of human colon cancer, this signalling event is also initiated by hyper‐expressed p38 that led to increased c‐Jun synthesis, MMP‐9 transcription, and MMP‐9‐dependent invasion through p38 interacting with c‐Jun. Thus, MAPK may act as both an activator and a cofactor of transcription factors to regulate MMP expression, finally leading to an
“invasive” response (77).
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Others have suggested that the combination of p38 and JNK are the most important regulatory signalling unit influencing MMPs expression and that both are required for the synergistic induction of MMPs by cytokines and growth factors (78). Indeed, other pathways have already been shown to regulate MMP‐9 expression individually (79) such as the ERK pathway which is mainly activated by growth factors and has been linked to cell proliferation, cell growth, and differentiation, and contributes to the transcriptional regulation of MMP‐9 in arterial smooth muscle cells (78).
Fig. 1.5: Signaling pathways involved in MMP gene transcription, and potential strategies for therapeutic intervention (according to Overall & Lopez‐Otin 2002 (11), modified. Compounds that are able to block the transcription of matrix metalloproteinase (MMP) genes at different levels are shown in red boxes. Extracellular
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factors, such as interferon‐ (IFN‐ ) inhibit MMP transcription via the JAK–STAT signalling pathway. Monoclonal antibodies against tumour necrosis factor‐ (anti‐TNF), soluble forms of the TNF receptor (sTNF‐R), natural antagonists of the interleukin (IL)‐1 receptor‐ >(IL‐1R ) or soluble forms of this receptor (sIL‐R) can block signalling pathways initiated by extracellular factors such as TNF‐ and IL‐1, which induce MMPs in cancer cells. Compounds such as manumycin A, SB203580, malolactomycin, SP600125, or PD98059 act at different levels to block the signal‐transduction pathways that are associated with MMP transcriptional induction in human tumours. Finally, there are several possibilities to target the nuclear factors that are responsible for MMP transcriptional up‐
regulation. Glucocorticoids, terpenoids, curcumin, nobiletin or NSAIDs (non‐steroidal anti‐inflammatory drugs) block the activity of transcription factors such as AP1 and NF‐ B, which regulate the transcription of several MMP genes.
Similarly, restoring the activity of transcription factors such as p53 and TEL, which negatively regulate MMP expression and the activity of which is lost in human tumours, could down regulate these genes. IFN‐ , interferon‐
; I B, inhibitor of B ; I BK; inhibitor of B kinase; JAK, JUN‐activated kinase; MAPK, mitogen‐activated protein kinase; MAPKK, mitogen‐activated protein kinase kinase; MAPKKK, mitogen‐activated protein kinase kinase kinase;
NF‐ B, nuclear factor of B; STAT, signal transducer and activator of transcription; TEL, translocation‐ETS‐ leukaemia.
1.7.2.3 Transcription Factors
The analysis of a 600 bp fragment of the MMP‐9 promoter in human shows several potential TF‐binding sites responsible for its regulation (80). As shown in Fig. 1.6, the promoter of the MMP‐9 gene mainly contains BS for NF‐κB, Sp1, AP‐1, and other TF (e.g. C/EBP, PEA3) (81, 82).
These binding sites are highly conserved in humans, rats, and mice.
Fig. 1.6: Regulatory elements in the promoter regions of MMP‐9. (according to Overall & Lopez‐Otin 2002 (11), modified). The promoters are shown in the direction 5'–3', with the transcription start sites indicated with a bent arrow, and the transcription‐factor‐binding sites placed within boxes. The relative positions of the different elements are not drawn to scale. Transcription‐factor‐binding sites include: the activator proteins (AP)‐1, the retinoblastoma control element (RCE), the keratinocyte differentiation‐factor responsive element (KRE), the transforming growth factor b inhibitory element (TIE), nuclear factor of B (NF‐ B) site, the polyomavirus enhancer‐A binding‐protein‐3 (PEA3) site, the TATA‐box (TATA).
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1.7.2.4 Activators and inhibitors
On the protein level, MMP‐9 zymogen is also activated via the “cysteine switch” in which the cysteine‐zinc interaction is abrogated by removal of the pro‐peptide domain (83). Upon activation, MMP‐9 is converted from a 92 kDa zymogen form to an 82 kDa active form (84).
MMP‐9 is activated by other MMPs, including MMP‐2, ‐3, ‐13, ‐17, and ‐26 (85‐88). For example, plasmin/MMP‐3 mediates an activation mechanism by which plasmin or MMP‐3 directly activates pro‐MMP‐9 (89) (Fig. 1.7).
Further regulation of MMP activity is offered by the TIMPs. Inhibition of MMP‐9 activation is performed by TIMP‐1 binding to the zymogen forms of the enzyme (90). Interestingly, it has been shown that TIMP‐1 is also able to bind and to inhibit the activity ofmature MMP‐9 (40, 43).
However, already activated MMP‐9 proteins are mainly controlled by TIMP‐3 (90, 91).
In circulation, α2macroglobulin inhibits MMP‐9 to prevent systemicMMP‐9 activation (29).
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also regulate inflammatory processes, e.g., by processing IL‐1β to its biologically active and mature form (95). It has also been suggested to play a role in the apoptotic process (96).
Additionally, MMP‐9 plays an important role in angiogenesis and neovascularization (97). MMP‐
9 has been shown to be importantly required for the recruitment of endothelial stem cells, a critical component of angiogenesis (97). Consequently, KO models of MMP‐9 resulted in delayed vascularization in addition to effects like apoptosis (98). Other studiesin MMP‐9 KO indicated that MMP‐9 plays a key role in cardiac rupture after myocardial infarction (99).
Moreover, neutrophil‐derived MMP‐9 appears to be elevated during acute myocardial infarction (MI), whereas macrophage‐secreted MMP‐9 is more important during subsequent tissue remodeling. Thus, MMP‐9 plays an important role in ventricular remodeling after acute MI and it has been concluded that plasma levels of MMP‐9 following MI may be predictive for this process.
High levels of MMP‐9 in patients with coronary artery disease have been recently reported (100) and high MMP‐9 levels also correlate with coronary artery ectasia (101) thus also serving as a predictor of increased mortality in patients with coronary artery disease (102). Serum levels of MMP‐9 have been reported to be elevated inpatients with MI and angina (103). Moreover, MMP‐9 plays an important role in animal models of both cerebral ischemia and human stroke. A critical role of MMP‐9 has been also shown for the development of abdominal aortic aneurysm using MMP gene deletion mice (104). Since MMP‐9 cleaves a variety of ECM molecules, recent studies on atherosclerotic plaque stability using a series of apoE/MMP‐9 double KO mice have indicated that MMP‐9 has a protective role by limiting plaque growth and enhancing plaque stability (105).
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1.9 MMP‐9 as a biomarker
Biomarkers are precious allies for research in the field of in vitro diagnostics which can be used to diagnose and monitor aetiology and progression of diseases and guide therapeutic decisions (106). Many clinical studies have established a relationship between MMP‐9 and certain diseases, making MMP‐9 a feasible candidate to add to the multiple biomarker lists. For example, a recent study has established a relationship between MMP‐9 and post‐MI remodeling and mortality thus making MMP‐9 a proximal biomarker for cardiac remodeling and a distal biomarker for inflammation (107). Additionally, some other studies showed the relationship between MMP‐9 and different types of cancers. For example, MMP‐9 levels in serum, plasma, and urine are significantly elevated in patients with breast cancer (108) and stage III or IV lung cancer (109). Urinary MMP‐9 levels also correlate with presence stage, and grade of bladder cancer (110). In addition, serum levels of MMP‐9 are significantly higher in patients with pancreatic ductal adenocarcinoma than in healthy controls (111). Both latent and activated forms of MMP‐9 have been detected in the cerebrospinal fluid of patients with brain tumors (112). Moreover, salivary MMP‐9 could be considered as a sensitive and specific diagnostic &
prognostic biomarker in the detection of oral lichen planus (OLP) (113).
It is important to note that the measurement of MMP‐9 in body fluids, in particular serum or plasma, can be influenced by the type of fluid and method of collection and storage. For example, basal MMP‐9 levels in serum/plasma can be influenced by the use of EDTA or heparin (114), a problem that can be alleviated by using sodium citrate instead (115). Another issue to be considered is that of sample storage. For example, it has been reported that plasma MMP‐9 is unstable and degrades rapidly even when stored at −80°C (116).
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However, the influence of direct and indirect effects of different anti‐coagulants and their impact on MMP‐9 expression is still poorly elucidated. Thus, further studies are needed to assess MMP‐9 as a biomarker due to controversial results and non‐standardized procedures in pre/post‐analytical.
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1.10 Aims of the study:
In this study, the effect of different anticoagulants on the regulation of MMP‐9 will be characterized, including the identification of molecular mechanism mediating these effects to assess the impact of the respective substances on the suitability of MMP‐9 as a biomarker.
The specific aims of the present study are:
To identify the effect of different anticoagulants on MMP‐9 expression. To identify the major MMP‐9 expressing cell type(s) in the blood.
To identify the mediator(s) inducing MMP‐9 production.
To elucidate the molecular mechanism(s) regulating MMP‐9 expression.
To characterize further cellular effects shown by the respective cell type(s) under MMP‐9‐ inducing conditions.
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2 Materials
2.1.1 Chemical Reagents:
Reagents Company/Origin
Acrylamide solution Roth, Karlsruhe, D
Agar Gibco Life Technologies, Karlsruhe, D
Agarose Biozym, Oldendorf, D
Ampicillin Sigma Aldrich, Darmstadt, D
Bovine serum albumin, fatty acid free Sigma Aldrich
Cell culture media: RPMI Gibco Life Technologies
Ethidium bromide Sigma Aldrich
EDTA Sigma Aldrich
Fetal serum albumin Biochrome, Berlin, D
Glycine Merck, Darmstadt, D
PBS buffer (pH 7.4) Biochrome
5x passive lysis buffers Promega, Heidelberg, D
Heparin Ratio pharm, ULM, D
Citrate Sarstadt, Numbrecht, D
Low molecular weight heparin Sanofi, Paris, France
2.1.2 Enzymes
Enzymes Company/Origin
Taq DNA‐Polymerase Qiagen, Hilden, D
Superscript ™ II Reverse Transcriptase Invitrogen, Karlsruhe, D
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2.1.3 DNA size markers
DNA size markers Company/Origin
100 base pair ladder Invitrogen, Carlsbad, California, USA
1 kilobase pair ladder Invitrogen
2.1.4 Laboratory Equipment
Laboratory Equipment Company/ Origin
Agarose Gel electrophoresischamber Pharmacia, Erlangen, D
Autoclave ELV 3870 Sartorius, Göttingen, D
Disposal Pasteur pipettes Heraeus, Hanau, D
Disposable syringes ( 1ml , 2ml, and 10ml ) Brand, Wertheim, D
Electrophoresis Chamber Scotsman, Vermon Hills, USA Reaction tubes: 0.2, 0.5,1.5, 2 ml Sarstedt, Frickenhausen, D Filter paper for Western blots Schleicher & Schnell, Dassel, D
Freezer (‐20 ° C) Einzelhandel, D
Freezer (‐80 ° C) Einzelhandel, D
Refrigerator (Liebherr) Einzelhandel, D
Refrigerated centrifuge (Thermoscientific) Sorvall, Bad Homburg, D
Culture shakers Brunswick scientific, Cambridge, UK
Light microscope (Primovert) Zeiss, Oberkochen, D
LightCycler480 Roche Diagnostic, Basel, Schweiz
Luminometer (Lumistar) BMG lab technologies, Offenburg, D Microbilogical incubator (BD: 17053) WTB Binder, Tuttlingen, D
Microtiter plates, 96‐well, round bottom Greiner, Frickenhausen, D
Microwave Einzelhandel, D
PCR Gradient cycler (Biometra) Biometra, Göttingen, D
Petri dishes Corning, Corning, USA
Pipettes Abimed, Langenhagen, D
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Pipette tips Sarstedt
Pipetting Brand
Ultrapure water (Mili‐Q) Millipore Merck, Darmstadt, D
Sterile workbench Heraeus
Thermocycler (Biometra) Biometra
2.1.5 Buffers
50 x TAE‐Stock solution
Tris base 242 g
0.5M EDTA 100 ml
solution of glacial acetic acid 57.1 ml, then fill to 1 liter with distilled water 1 x TAE buffer (+ ethidiumbromide)
50x TAE stock solution 20 ml
distilled water 980 ml
ethidium bromide 40 μl
Trypsin / EDTA solution
0.5% (v / v) trypsin 0,25 ml
0.02% (v / v) EDTA 0,10 ml
PBS 500 ml
1x PBS solution
NaCl 10 g
KCl 0,25 g
Na2HPO4 1,41 g
KH2PO4 0,3 g
Distilled H20 1000 ml; adjust pH 7,4
TBS (10x)
Tris base 24,2 g
NaCl 80 g
Distilled H20 1000 ml; adjust pH 7,6
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1X wash buffer
25x wash buffer 40 ml
Distilled H2O 960 ml
3.7% Buffered Formaldehyde
37% Formaldehyde 5 ml
10 x PBS 5 ml
Distilled H2O 40 ml
1x TdT Labelling Buffer
10X TdT Labelling buffer 5ml
Distilled H2O 45 ml
1x TdT Stop Buffer
10X TdT Stop buffer 5ml
Distilled H2O 45ml
2.1.6 Commercial Kits
Commercial Kits Company/ Origin
Agarose gel extraction kit Qiagen
Rneasy Mini Kit Qiagen
Proteome Profiler Human XL cytokine Array R&D systems, Weisbaden, D
kit
CytoSelect Phagocytosis Assay kit Cell Biolabs, San Diego, California, USA
Via‐Light Plus kit LONZA, Nottingham, Ireland
TACS TdT kit TREVIGEN, Gaithersburg, USA
2.1.7 Software
Software Company/ Origin
Light Cycler 480 release 1.50. SP4 Roche, Basel, Schweiz
Tool Lab TL100 Sigma‐Aldrich, Saint Louis, Missouri, USA
Image J NIH, Bethesda, Maryland, USA
Apoptosis Olymbus Italia, Segrate, Italy
Nano drop Thermo Fisher Scientific, Waltham, Massachusetts,
29
USA
Phagocytosis Cell Biolabs, San Diego, California, USA
Power point 2010 Microsoft , Redmond, Washington, USA
Endnote X7 Thomson Reuters Scientific, Philadelphia, USA
Graph Pad Prism 6 Graph Pad Software, La Jolla, California, USA
Microsoft Office 2010 Microsoft, Redmond, Washington, USA
Photoshop 8 Microsoft, Redmond, Washington, USA
2.2 Methods
2.2.1 Blood Specimen ‐ Collection and Storage
A total of 20 venous blood samples were collected in different commercially available monovette tubes (Sarstedt, Germany) containing either ethylene‐diamine‐tetra‐acetic‐acid (EDTA), lithium‐heparin (heparin), or sodium citrate, from donors (female: 9, male: 11; mean age ± SD = 30 ± 9 years) who presented at the Department of Clinical Chemistry at Hannover Medical School between August 2013 to August 2014. Blood samples were incubated on a rolling plat form for 30 min at RT, then centrifuged at 1900 × g for 10 min, and after removal of the supernatant, serum and plasma samples were aliquoted and immediately used or stored at− 80 °C until assay.
Medical Ethics in the research study was considered under an informed consent that was given by all blood donors before inclusion. The blood collection was performed by Bernadette Lüns.
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2.2.2 Cell Culture 2.2.2.1 Cell lines
To analyze the influence of different anticoagulants on MMP‐9 expression, 3 different cell lines were used: THP‐1 cells (which are derived from a patient suffering from acute monocytic leukemia), Jurkat cells (an immortalized line of human T‐lymphocytes), and HT cells (purified B‐
cell lymphoma cells; all from DMSZ, Germany). All cells were cultured in the RPMI 1640 medium containing 300 mg/L L‐glutamine (PAA, Linz, Austria), supplemented with 10% fetal calf serum (FCS, Biochrom, Berlin, Germany), without antibiotics at 37°C in a humidified atmosphere containing 5% CO2. All THP‐1, Jurkat, and HT cells in single, double, and triple co‐cultures were stimulated by HMWH, LMWH, EDTA, and citrate in dose and time dependent experiments. Some of these experiments were performed by Bernadette Lüns.
2.2.2.1.1 Individual cultures
Individual cultures of monocytes (THP‐1), T‐cells (Jurkat), or B‐cells (HT) were incubated with HMWH, LMWH, EDTA, or citrate. 2 x 106 cells / well (2 ml of medium in 6‐well plates) were cultivated (10% FCS) or starved (1% FCS) overnight and then stimulated up to 24h with 3.2 mg EDTA, 10 µl heparin (= 50 IU), or 220 µl citrate in a time dependent experiment.
Following stimulation, RNA was isolated and cDNA was synthesized according to manufacturer’s instructions (see 2.4.1, 2.4.3). Afterwards, the mRNA expression of MMP‐9 was analyzed (see 2.5.2). Supernatant of stimulated cells were stored for further analysis with ELISA (see 2.6.1) for MMP‐9 secretion or with the Proteome Profiler Human XL Cytokine/Chemokine Array for the detection of secreted mediators (see 2.7.1).
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2.2.2.1.2 Stimulation of individual monocytes cultures with T‐cell‐derived supernatant
Furthermore, 2 x 106monocytes / well (2 ml of medium in 6‐well plates) were stimulated with the supernatant of anticoagulant‐treated T‐ or B‐cells, i.e., THP‐1 were stimulated with the supernatant of HMWH‐ (50 IU), citrate‐ (220 µl), or EDTA‐ (3,2mg/well) treated Jurkat or HT cells (pre‐starvation phase: overnight, anticoagulant treatment duration: 24h). Starved monocytes (1% FCS overnight) were incubated for 0, 2, 4, 6, and 24h with the supernatant of anticoagulant‐stimulated cells and then treated as described previously (see 2.2.2.1.1).
2.2.2.1.3 Stimulation of individual monocyte cultures with the supernatant of T‐cells stimulated with derived human plasma derived from heparin monovettes
Moreover, 2 x 106monocytes / well (2 ml of medium in 6‐well plates) were cultivated (10% FCS) or starved (1% FCS) overnight and then incubated with supernatant from T‐cells stimulated with human plasma derived from heparin containing monovettes (pre‐starvation phase: overnight, treatment with human heparin plasma: 24h). Starved monocytes (1% FCS overnight) were incubated for 0, 2, 4, 6, and 24h with the respective T‐cell supernatant and then treated as described previously (see 2.2.2.1.1).
2.2.2.1.4 Double co‐culture experiments
To assess the influence of the interaction of different cell types on MMP‐9 expression, double co‐culture experiments were performed by which monocytes or T‐cells (THP‐1 and Jurkat), monocyte and B‐cells (THP‐1 and HT cells), or T‐cells and B‐cells (Jurkat and HT cells) were incubated together. 1 x 106 cells / well per cell line (= 2 x 106 cells / well) were cultured in 2 ml medium in 6 well plates. Following starvation (1% FCS overnight), cells were stimulated with the
32
respective anticoagulant as described previously(see 2.2.2.1.1) incubated for 0, 2, 4, 6, and 24h and then treated as described before (see 2.2.2.1.1).
2.2.2.1.5 Triple co‐culture experiments
To further analyse the effects of interactions between different cells types, triple co‐culture experiments were performed, i.e., a mixture of all three cell lines (THP‐1, Jurkat, and HT cells) were performed (see 2.2.2.1.1). 7 x 105 cells / well and per cell line (= 2.1 x 106 cells / well) were starved overnight in 2 ml of medium (1% FCS) in 6‐well plates, then incubated with the corresponding anticoagulants for 0, 2, 4, 6, and 24h, and finally treated as described above (see 2.2.2.1.1).
2.3 Anticoagulants
2.3.1 High molecular weight heparin (HMWH), a highly sulfated glycosaminoglycan known to bind to the hemopexin domain of MMPs, is often found associated with MMPs on the cell surface. Due to its high negative charge density, HMWH prevents clotting and is widely used in blood sampling (117, 118). For individual, double, or triple co‐culture experiments, stimulation with 50 IU HMWH was performed.
2.3.2 Low molecular weight heparin (LMWH), a new class of anticoagulant derived from unfractionated heparin (UFH), has an advantage over HMWH by which it is easily distributed due to its low molecular weight. This has led to its increasing use for a number of thromboembolic indications. For individual or double co‐culture experiments, stimulation with 50 IU Clexane or Fragmin was performed.
33
2.3.3 Ethylene diamine tetra acetic acid (EDTA), a widely used anticoagulant due to its ability to
"sequester" metal ions such as Ca2+ and Fe3+. After being bound by EDTA, metal ions remain in solution but exhibit diminished reactivity. In coagulation studies, EDTA has the role to inhibit the clotting process by removing calcium from the blood. Furthermore, it is also known to inhibit a range of metallopeptidases via the chelation of the metal ion required for catalytic activity (119).
For individual, double, or triple co‐culture experiments, stimulation with 3.2mg/ml EDTA was performed.
2.3.4 Citrate, a chelating agent, is used to chelate calcium ions and therefore inhibits coagulation (usually in the form of tri‐sodium citrate). For individual, double, or triple co‐culture experiments, stimulation with 220µl/well citrate was performed.
2.4 RNA isolation and cDNA synthesis 2.4.1 RNA isolation and purification
For RNA isolation and purification, cells were harvested as a cell pellet and an appropriate volume of Buffer RLT was added. Afterwards, 1 volume of 70% ethanol was added to the lysate and mixed well by pipetting. The sample was transferred to an RNeasy Mini spin column placed in a 2 ml collection tube and centrifuged for 15 s at ≥ 8000 x g. After discarding the flow, 700 μl Buffer RW1 were added to the RNeasy spin column and centrifuged. After discarding the flow, two times 500 μl Buffer RPE were added to the RNeasy spin column and centrifuged for 2 min.
Finally, RNA was eluted in 50 μl RNase‐free H2O and concentrations were determined using the Nano‐drop ND‐1000 photometer.
34
2.4.2 RNA Quantification Using Nano‐Drop ND ‐1000
To check the concentration and quality of RNA samples, 1 μl of each sample was analyzed using the Nano Drop ND‐1000 spectrophotometer according to the manufacturer's instructions. At a wavelength of 260 nm, optical density of the sample was determined (reference against dH2O or the respective buffer). To check the purity of the RNA, the ratio of the absorption values at 260 nm and 280 nm was determined. In all cases the value of A260 / A280 ranged from 1.8 to 2, indicating the purity of the isolated RNA.
2.4.3 cDNA synthesis
Total RNA (1 μg) was reverse transcribed using the Superscript™ II Reverse Transcriptase kit (Invitrogen, USA) as described in the manual, in which 2 μl of random primer and 2 μl of nucleotides were incubated for 5 min at 65 °C, then 10 min at 25 °C, 50 min at 42 °C, and 15 min at 70 °C. Finally 60 μl of t‐RNA were added to fill to a total volume of 100 μl. RNA isolation and cDNA synthesis were performed in part by Bernadette Lüns.
2.4.4 Agarose gel electrophoresis
For the separation of nucleic acid according to size, agarose gel electrophoresis was applied. For 1% or 2% agarose gels, 2g or 4g Agarose were dissolved in 200 ml 1 x TAE buffer and heated in a microwave for 4 minutes. The dissolved agarose was poured into the gel chamber and allowed to cool down for 15 minutes. Then samples were mixed with loading buffer containing bromophenol blue and ethidium bromide (ratio buffer to sample: 1:4) and loaded on the gel. 5 μl of 1 kb DNA ladder were used. Electrophoresis was carried out at a voltage of 100 V (30 minutes) and DNA bands were visualized on the transluminator with UV light (302 nm).
35
2.5 Detection of mRNA expression
2.5.1 Gradient polymerase chain reaction
Gradient PCR was applied to identify the optimum temperature of each primer pair. With the gradient PCR, a wide range of temperatures can be covered which facilitates the detection of the optimum annealing temperature for each specific PCR reaction.
The procedure was pipetted as, presented in Table 2.
Tab.2: PCR reaction pipetting procedure
Reagent 50 μl reaction 20 μl reaction Final Concentrations
volume volume
H2O Add to 50 Add to 20
10 x buffer 10 4 1x
10 mM dNTPs 1 0,4 200μM
Forward Primer 2.5 1 0,5 μM
Reverse Primer 2.5 1 0,5 μM
Template DNA 5 2 0,5 μM
(diluted 1:10)
GC‐Buffer 2.5 1 5 %
Fast Start DNA‐ 0,5 0,2 0,02U/μl
Polymerase
Most gradient PCRs were carried out using a mean annealing temperature of 65 °C, thus testing a range of 55 to 75 °C. This allowed the determination of the optimal amplification temperature.
For primer pairs with different calculated annealing temperature, the mean was temperature was adjusted accordingly.
36
