IMMUNOEXPRESSION OF MATRIX METALLOPROTEINASES MMP 1, MM2, MMP 9 AND MMP14 IN EXTRAGENITAL ENDOMETRIOSIS AND EUTOPIC ENDOMETRIUM [303706]

IMMUNOEXPRESSION OF MATRIX METALLOPROTEINASES MMP 1, MM2, MMP 9 AND MMP14 IN EXTRAGENITAL ENDOMETRIOSIS AND EUTOPIC ENDOMETRIUM

Eugeniu Cazacu1, Eremei Zota1, Claudiu Margaritescu2, Radu Niguleanu1, Ruslan Pretula1

1[anonimizat]

2[anonimizat], Romania,

Department of Pathology

Background: Matrix metalloproteinases are proteolytic enzymes responsible for the disorder of extracellular matrix modeling in endometriosis and their involvement in the invasion process. [anonimizat]1, MMP2, MMP9 and MMP14 [anonimizat].

Material and methods: The study included 40 female patients diagnosed with extragenital endometriosis. The used methods consisted in processing the specimens by classical histological technique with paraffin inclusion and enzymatic immunohistochemical technique for the detection of metalloproteinases MMP1, MMP2, MMP9 and MMP14.

Results: The expression of matrix metalloproteinases MMP2, MMP14 [anonimizat]9 was evident in both stromal and glandular cells in these lesions. The expression MMP1 was not present. Normal endometrial tissue showed high reactivity for MMP14 and low reactivity for MMP2 and MMP9.

Conclusions: This study reveals some aspects related to the morphological and clinical features of extragenital endometriosis with different locations and the correlation between the clinical evolution and some immunohistochemical markers with potential prognosis regarding the aggressiveness of such lesions.

Key words: Endometriosis, [anonimizat] a comon, benign, inflamatory, pathology, represented by the ectopic location of functional endometrial glands and stroma outside the uterine cavity[1,2].

[anonimizat]-de-sacs, the uterine ligaments and the pelvic wall. [anonimizat], [anonimizat]. [anonimizat], the lungs or even the brain. [3]

The prevalence rate of symptomatic endometriosis is estimated to be 10% with an incidence of about 2-7/1000 women per year and a further 11% [anonimizat] a [anonimizat], [anonimizat] a transient phenomenon [4]. Endometriosis is associated with a [anonimizat], [anonimizat] a strong adverse effect on the physical and mental health of the patient. While endometriosis is assumed to be a [anonimizat] a certain resemblance to malignant tumors due to the characteristic of infiltration. While the development of endometriosis has been shown to be a complex process involving interaction between genetic and environmental factors, the etiology and pathology of endometriosis remain poorly understood [5].

Numerous theories were proposed to explain etiology of endometriosis.

Matrix metalloproteinase (MMP) represents a large family zinc-dependent endopeptidases, involved in the degradation of the extracellular matrix in the process of endometrial cell implantation and are classified by their substrate specificity [6,7]. Several subtypes of MMPs are distinguished, depending on their substrate-specificity and localization: collagenases, gelatinases, stromelysins, matrilysins and membrane-type metalloproteinases. MMPs play a crucial role in numerous physiological processes, for instance: bone remodeling, angiogenesis, inflammation, ovulation and embryogenesis. What is more, MMPs are involved in cyclic changes of endometrium structureand thickness in the steroid hormones concentration levels. MMPs are expressed in both epithelial and stromal cells.

Numerous MMPs are additionally involved in many pathological processes, such as: fibrosis, weakening of matrix (e.g., in aortic aneurysm or dilated cardiomyopathy) or tissue destruction (e.g., caner invasiveness, also endometrial carcinoma invasiveness, and ability to metastasize) [8].

Matrix metalloproteinases (MMPs) are essential in orchestrating proper physiological functioning of the endometrium; hence, alteration of MMP activities is considered as a critical factor for the development of endometriosis. MMPs are involved in the cellular event of epithelial-mesenchymal transition [9].

Objective of the research was to determine the activity of MMP1, MMP2, MMP9 and MMP14 in endometriotic lesions of woman with endometriosis and compare with normal endometrium.

Material and methods

The study group included 40 female patients diagnosed with extragenital endometriosis from 2010 – 2017, diagnosed and surgically treated at Department of Surgery, Obstetrics and Gynecology from “Gherghe Paladi” Institutia Medico-Sanitara Publica Spitalul Clinic Muicipal, “Sfântul Arhanghel Mihail” Institutia Medico-Sanitara Publica Spitalul Clinic Muicipal, Chisinau, Republic of Moldova and Emergency County Hospital, Craiova, Romania. Endometrial samples normal proliferative endometrium from woman who underwent hysterectomy for benign conditions were also included for comparison. The investigation was performed by using the archival paraffin-embedded tissue blocks corresponding to these cases, preserved at the Morphopathology Departments of hospitals. Hematoxylin and eosin slides ware reviewed by 3 pathologists to confirm the diagnosis. Pertinent clinical and demographic information was recorder for all study cases.

The research protocols was approved by the Ethics Committees of Nicolae Testemitanu State University of Medicine and Pharmacy, based on the patients informed written consent for the usage of the biologic material for research, after the pathological exam requested the diagnosis.

For immunohistochemistry, we used the paraffin blocks archived in the Laboratory of Pathology of the same Hospital. From these, 4 μm thick seriate sections were cut, which were further dewaxed, clarified and hydrated. Then, endogenous peroxidase was blocked with hydrogen peroxide (0.3%) for 15 minutes, at room temperature, and as antigen retrieval, we used microwaving for 20 minutes in 0.1 M citrate buffer of pH 6 (with the exception of fibronectin for which we used enzymatic retrieval with proteinase K, for 15 minutes, at 37°C). To avoid the nonspecific binding, the slides were covered with 2% bovine serum albumin (BSA) for one hour, at room temperature. Subsequently, the slides were incubated overnight, at 4șC, with the primary antibodies whose characteristics are presented in Table 1. Then, we used an amplification based on labeled Strep-tavidin–Biotin 2 (LSAB2) enzyme detection system and the correspondent Dako kit (Redox, Romania – K0675).

As chromogen, we used the 3,3’–Diaminobenzidine (DAB, Dako, K3468) and the Mayer’s Hematoxylin kit (Tunic, Bio-Optica, Romania – M06002) for counter-staining. As negative internal controls, we used the same slides and procedures, but omitting the primary antibody.

Table 1 Antibodies used in immunohistochemical study

The interpretation of the immunohistochemical reactions aimed first of all at highlighting the chromogen at the level of the antigenic targets and then at quantifying both the intensity of this signal and the proportion of the immunoreactive cells. The intensity of the reactions was graded using a scale with 4 degrees: 0 for the absence of reactivity; 1 for a light intensity; 2 for moderate intensity and 3 for high intensity (intense reaction). The reactivity of the inflammatory infiltrate (neutrophils, macrophages) was used as internal control to evaluate the intensity of these markers, this being considered the maximum reactivity.

A personalized score has been used to evaluate MMP1, MMP2, MMP9 and MMP14 expression: the percentage of tumoral positive cells (P) and the intensity of staining (I), obtaining a P×I final score.

The proportion of positive cells was also estimated on a 4-degree scale: 0 no positive cells in any microscopic field; 1, <10% of positive cells; 2- between 10-50% of positive cells and 3> 50% of positive cells (the examination being done at the x40 objective, respectively on 10 fields). The final score (SIHC) was obtained by multiplying the 2 scores, respectively the qualitative one (intensity) and the semiquantitative one (the proportion of reactive cells) and was graded as follows: negative (-) for 0; weakly positive (+) for 1; moderately positive (++) for 2; and intensely positive (+++) for scores 3. In positive immunochemistry, the maximum score was 9 and minimum–1. In our score system, we established a threshould value of 4, considering a value >4 as a high score, and a value ≤4 as a low score.

Results

The patient group consist 42 women with endometriosis, age range of 21-63 years (median 40). Location included: the anterior abdominal wall after caesarean operation–20, inguinal hernia–7, umbilical hernia–4, perineal region–1, appendix–4, colon–5, and ileum–1case. The classical histopatological examination of the general expression of MMP1, MMP2, MMP9 and MMP14 revealed a diverse variability. For MMP2, MMP9, MMP14 markers, the reaction positivity was variable not only from one case to another but also within each case, the latter being characterized by the identification of different areas within endometriosis. Consequently, the complex staining patterns was homogenous and heterogenous, as a refletion of the endometriotic cells equal and variable reaction capacity in diferent neighboring teritories. We should also noted that the glandular cell deposit in the center expressed the investigated molecules compared to a weak expression in the peripheral focus of endoetriosis.

Our data resulting from the immunohistochemical evaluation of MMP1, MMP2, MMP9, MMP14 respectively, based on the quantification of immunopositive tumoral cells count and staining intensity, and expressed as individual scores, are sumarized in Tables 2.

Table 2 – Characteristics MMP1, MMP2, MMP9 and MMP14 immunoexpression of endometriotic tisue and eutopic endometrium.

The expression of matrix metalloproteinases MMP2, MMP14 was significant in glandular cells from endometriotic lesions, while MMP9 was evident in both stromal and glandular cells in these lesions. The expression MMP1 was absent in wall cases of endometriosis and in normal tissue. Normal endometrial tissue showed high reactivity for MMP14 and MMP9 and low reactivity for MMP2.

The reactivity of MMP2, MMP9 and MMP14 metalloproteinase in endometriotic lesions was higher than in eutopic endometrium. MMP2 and MMP9 IHC staining were performed, and endometriosis – associated fibrosis and lymphocytic infiltrate evaluated with MMP1. Nuclear staining of MMP2 for glands and both membranous and cytoplasmic staining of MMP9 for stroma were considered positive. In cases with intestinal endometriosis staining was diffuse and intensity was strong (3+), in abdominal wall endometriosis intensity was moderate (2+) while eutopic endometrium staining was diffuse and intensity was weak to moderate (1-2+) in all cases. MMP14 was positiv and intensity was moderate in both cases. The expression of MMP1 on the examined tissues, was absent in endometriosis and in cases of eutopic tissue.

Immunohistochemical analysis has demonstrated the significant enhance of MMP9 and MMP14 expresission in endometriosis and in endometrium. The distinctive feature of MMP9 and MMP14 expression in endometriosis was considerable increase of its activity precisely on the border of endometriotic lesion and the peritoneum. Elevation of MMP9 and MMP14 activity was also observed in the stroma of the ectopic endometrium bordering on the underlying stroma (Figure 3, E, F, G, H). Enhanced activity of MMP9 in the stroma of the ectopic endometrium bordering on the underlying stroma was accompanied by the formation of macrophage-lymphocyte infiltrates, (figure 3, E), that evidenced the implication of immune cellular components into the inflammation zone. Thereby, the obtained reaserch findings allow to suggest, that elevation of MMP9 activity in the sites of endometriotic lesion on the border with the underlying stroma promotes the invasiviness of ectopic endometrium by remodeling of the underlying stroma and infiltration of endometrial cells into the peritoneum. Increased activity of MMP2 was observed in stroma of the ectopic endometrium bordering on the underlying stroma as well (figure 3, C). The intensified MMP9 expression in normal endometrium was found to prevail in outer membranes of endometrial cells (figure 3, F) while than expression of MMP14 was found in intern. Thus, the obtained findings are indicative of the increased MMP9 and MMP14 activity in the sites of endometriotic lesion.

Endometriosis Normal endometrium

Table 3. A, C, E, G- MMP1, MMP2, MMP9 and MMP14 expression on ectopic endometrium of women with endometriosis. B, D, F, H – MMP1, MMP2, MMP9 and MMP14 expression on normal endometrium.

Discusion

Endometriosis i sone of the most common diseases affecting women of reproductive age. Thus far, the pathogenesis of this disease remains poorly understood. Endometriosis is considered a benigne disease that nontheless has the property of tissue invasion [5].

Metalloproteinases, enzymes that are important for extracellular matrix turnover, have recently been implicated in invasion and development of endometriosis. MMPs appear to be overexpressed in endometriotic lesions and contribute to establishment of endometrial glands and stroma at ectopic sites [10, 11].

MMP-1 (interstitial or fibroblast collagenase or colagenase-1) is the major collagenase able to degrade native fibrillar collagens type III, I, and II, at a specific site three –fourths from the N-terminus. MMP-1 is produced by various types of cells: fibroblasts, keratinocytes, endotelial cells, macrophages, hepatocytes, chondrocytes and asteoblasts [4 Biology of metallo].

MMP-2 (gelatinase-A, 72-kDa type IV collagenase) cleaves collagen type IV, the major BM constituent, as well as degraded collagen and some noncollagenous ECM glycoproteins. MMP-2 also degrades native type I collagen. TNF-α and β stimulate MMP-2 production and early conceptus (blastocytes) and IFN-τ repress MMP-2 production. MMP-2 is expressed by various cell types, including fibroblasts, keratinocytes, osteoblasts, and monocytes .

MMP-9 (gelatinase-B, 92-kDa gelatinase) cleaves N-terminal telopeptide of type I collagen in an acidic environment, playing a role in the remodeling of collagenous ECM. MMP-9 is produced by normal alveolar macrophages, polymorphonuclear leukocytes, osteoclasts, keratinocytes, and invading trophoblasts.

MMP-14 (membrane type-1-matrix metalloproteinase) collagen I, II, III, α-1 microglobulin, α-2 macroglobulin, (MT1-MMP) α-2-HS-glycoprotein, α1-antitrypsin, α1-proteinase inhibitor, α5 integrin, apolipoprotein A, apolipoprotein E and apolipoprotein J, brain-specific angiogenesis inhibitor 1, casein, CD44, fibrin II, E-cadherin, elastin, endoglin, entactin, epidermal growth factor receptor, extracellular matrix metalloproteinase inducer, gelatin, fibrillin, fibrin, fibrinogen, fibroblast growth factor recetor (FGFR) 1 and 4, fibronectin, galectin-3, gelsolin, growth differentiation factor-1, heparin-binding epidermal growth factor, hepatocyte growth factor activator inhibitor-1, inter-α inhibitor H4, intercellular cell adhesion molecule-1, kidney injury molecule-1, Kisspeptin/metastin, laminin, mannose-binding lectin, mucin 1, N-cadherin, Notch1, pro-MMP-2, -8 and -13, pro-transforming growth factor β, progelatinase A, receptor-activator of NF-kB ligand, stromal cell-derived factor 1, testican-1, transforming growth factor-β, transglutaminase, vitronectin.

The presence and role of MMPs in endometriosis is unquestionable, however the exact sequence of events is unclear. It remains obscure, whether changes in their activity occur primarily in uterine cavity, that due to cellular memory allows endometrium fragments implantation into peritoneum, or whether specific conditions in peritoneal cavity (inflammation or other immunological processes) change MMPs activity in endometrial cells discarded from uterus during retrograde menstruation. Moreover, some balance switches in matrix remodeling in eutopic endometrium can be periodic or temporal and vanish just after lesion constitution on ectopic site. What is more, changes of MMPs, demonstrated in uterine endometrium from women with endometriosis may also occur after implantation of ectopic lesions, under the influence of processes taking place in peritoneum. Solution of this riddle seems to be hard to find, as it would require prospective study based on endometriotic biopsies in asymptomatic population of women and long-term observation for endometriosis onset [12, 19].

In the literature, there is no agreement on differences in the expression of MMP-2 among endometriotic tissues and eutopic endometrium from women affected or unaffected by endometriosis. However, endometriotic tissues seem to express more MMP-2 than do eutopic endometrium [14, 15, 16, 17, 18].

Conclusions

The MMP 9 and MMP 14 activity significant elevation, established on ectopic endometrium of women with endometriosis. Study of MMP1, MMP2, MMP9 and MMP14 activities in endometriotic lesions from women with endometriosis is perspective for further investigation in order to determine a possible role of matrix metalloproteinases in the development of invasiviness process in case of extragenital endometriosis.

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