Progesterone receptor modulator CDB-2914 induces extracellular matrix metalloproteinase inducer in cultured human uterine leiomyoma cells

Effects of progesterone receptor modulator CDB-2914 on the expression of the extracellular matrix (ECM) components were examined in cultured human uterine leiomyoma and myometrial cells. ECM metalloproteinase inducer (EMMPRIN), matrix metalloproteinases (MMPs), tissue inhibitors of MMP (TIMPs) and collagen levels were assessed by Western blot analysis, MMP activity assay and real-time RT-PCR. RNA interference (RNAi) of EMMPRIN was performed using small interfering mRNA. In cultured leiomyoma cells, CDB-2914 treatment at concentrations greater than or equal to 10(-8) M significantly increased EMMPRIN, MMP-1 and MMP-8 protein contents and MMP-1, MMP-2, MMP-3 and MMP-9 mRNA levels, and activity of MMP-1, MMP-2, MMP-3 and MMP-9 in the medium. TIMP-1 and TIMP-2 were significantly decreased at mRNA and protein levels by CDB-2914 treatment at concentrations > or =10(-7) M in these cells. CDB-2914 treatment decreased types I and III collagen protein contents. However, CDB-2914 treatment did not affect the ECM component expression in cultured myometrial cells. RNAi of EMMPRIN abrogated CDB-2914-mediated both induction of MMPs and reduction of TIMPs and collagens in cultured leiomyoma cells. These results suggest that CDB-2914 modulates the expression of EMMPRIN, MMPs, TIMPs and collagens in cultured leiomyoma cells without comparable effects on myometrial cells.     

Expression of metalloproteinases and its inhibitor in later stage of rabbit neointima development

Neointima formation after arterial de-endothelialization refers not only to smooth muscle cell (SMC) migration and proliferation, but also involves extracellular matrix (ECM) metabolism. Most studies regarding the role of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in neointima have focused on the early phase of vascular remodeling. In this study, we examined the expression of MMP and TIMP in rabbit aortic neointima at a relatively late stage of lesion development, between 4 and 12 weeks after initial de-endothelialization. Northern blot analysis revealed expression of steady-state MMP-9 mRNA was increased up to the 4th week and MMP-2 mRNA to the 12th week after de-endothelialization. In situ hybridization shown that MMP positive cells were predominantly distributed in arterial neointima. Expression of TIMP-1 mRNA was continuously up-regulated up to the 12th week and TIMP-1 positive cells, primarily SMCs, were also localized to the neointimal tissue. Alteration at mRNA level was accompanied by that at protein level, as assessed by SDS-PAGE zymography for MMPs and immunoblotting for TIMP-1. The profile of alteration at protein level correlated well with that at mRNA level. These data suggest that synthesis of MMPs and TIMP is a prolonged process and arterial SMC is a major source of MMP production in arterial neointima. Enhanced synthesis of MMPs and TIMPs at late stage of neointimal development may contribute to arterial ECM metabolism.     

Dynamic expression of mRNAs and proteins for matrix metalloproteinases and their tissue inhibitors in the primate corpus luteum during the menstrual cycle

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) may be involved in tissue remodelling in the primate corpus luteum (CL). MMP/TIMP mRNA and protein patterns were examined using real-time PCR and immunohistochemistry in the early, mid-, mid-late, late and very late CL of rhesus monkeys. MMP-1 (interstitial collagenase) mRNA expression peaked (by >7-fold) in the early CL. MMP-9 (gelatinase B) mRNA expression was low in the early CL, but increased 41-fold by the very late stage. MMP-2 (gelatinase A) mRNA expression tended to increase in late CL. TIMP-1 mRNA was highly expressed in the CL, until declining 21-fold by the very late stage. TIMP-2 mRNA expression was high through the mid-luteal phase. MMP-1 protein was detected by immunocytochemistry in early steroidogenic cells. MMP-2 protein was prominent in late, but not early CL microvasculature. MMP-9 protein was noted in early CL and labelling increased in later stage steroidogenic cells. TIMP-1 and -2 proteins were detected in steroidogenic cells at all stages. Thus, MMPs and TIMPs are dynamically expressed in a cell-specific manner in the primate CL. Early expression of MMP-1 is suggestive of a role in tissue remodelling associated with luteinization, whereas MMP-2 and -9 may contribute to later stage luteolysis. TIMP expression may control MMP activity, until declining at luteolysis.     

Matrix metalloproteinases and their inhibitors in the nasal mucosa of patients with perennial allergic rhinitis.

BACKGROUND: Allergic rhinitis and asthma show many similarities in their epithelial and inflammatory responses to allergens. However, one notable difference is that disruption and desquamation of the epithelium is a characteristic feature of asthma, whereas in perennial allergic rhinitis the epithelium is intact and thickened. One reason for this might be differing expression of matrix metalloproteinases (MMPs) or their inhibitors (TIMPs). There are few published data on the presence of MMPs or TIMPs in the nasal mucosa in rhinitis. OBJECTIVE: The purpose of this study was to investigate MMP and TIMP mRNA and protein in nasal mucosa from subjects with perennial allergic rhinitis and from nonrhinitic control subjects. METHODS: Biopsy specimens of nasal mucosa were taken from 10 well-characterized subjects with perennial allergic rhinitis and 10 nonrhinitic control subjects. MMP and TIMP mRNA was quantified through use of competitive RT-PCR, and protein was detected by means of Western blotting and ELISA. RESULTS: TIMP-1 mRNA and TIMP-2 mRNA were present in nasal samples, but there was no significant difference between the 2 groups. Only small amounts of MMP-1, -2, -3, and -9 mRNA were detected in the same samples. The corresponding proteins were detected by means of Western blotting. TIMP-1 protein and TIMP-2 protein were quantified in tissue homogenates; there was no significant difference between the 2 groups. CONCLUSION: Our studies have demonstrated the presence of large amounts of TIMP-1 and TIMP-2 mRNA and protein in nasal mucosa. There is no upregulation of MMPs or changes in TIMP expression in the nasal mucosa of patients with allergic rhinitis.     

Selective progesterone receptor modulator asoprisnil down-regulates collagen synthesis in cultured human uterine leiomyoma cells through up-regulating extracellular matrix metalloproteinase inducer

BACKGROUND: A recent clinical trial demonstrated that selective progesterone receptor modulator asoprisnil is effective in reducing uterine leiomyoma volume. We investigated the effects of asoprisnil in vitro on the expression of the extracellular matrix (ECM)-remodeling enzymes and collagens in cultured leiomyoma and matching normal myometrial cells. METHODS: The expression of extracellular matrix metalloproteinase inducer (EMMPRIN), matrix metalloproteinases (MMPs), tissue inhibitors of MMP (TIMPs) and collagens were assessed by western blot analysis. RESULTS: Untreated cultured leiomyoma cells had significantly lower EMMPRIN (P < 0.05), MMP-1 (P < 0.05) and membrane type 1-MMP (MT1-MMP) (P < 0.01) protein contents, but significantly higher TIMP-1 (P < 0.05), TIMP-2 (P < 0.01), type I (P < 0.05) and type III (P < 0.01) collagen protein contents compared with untreated cultured myometrial cells. Treatment with asoprisnil at concentrations > or =10(-7) M for 48 h significantly (P < 0.05) increased EMMPRIN, MMP-1 and MT1-MMP protein contents, and decreased TIMP-1 (P < 0.05), TIMP-2 (P < 0.01), type I (P < 0.01) and type III (P < 0.05 at 10(-7) M; P < 0.01 at 10(-6) M) collagen protein contents in cultured leiomyoma cells compared with control cultures. However, asoprisnil treatment did not affect the protein contents of ECM-remodeling enzymes and collagens in cultured myometrial cells. CONCLUSIONS: These results suggest that asoprisnil may reduce collagen deposit in the ECM of cultured leiomyoma cells through decreasing collagen synthesis and enhancing the expression of EMMPRIN, MMPs and TIMPs without comparable effects on cultured myometrial cells.     

Tension Force Downregulates Matrix Metalloproteinase Expression and Upregulates the Expression of Their Inhibitors through MAPK Signaling Pathways in MC3T3-E1 cells

Objective: Matrix metalloproteinases (MMPs), produced by osteoblasts, catalyze the turnover of extracellular matrix (ECM) molecules in osteoid, and the regulation of MMP activity depends on interactions between MMPs and tissue inhibitors of metalloproteinases (TIMPs). We focused on the degradation process of ECM in osteoid that was exposed to mechanical strain, and conducted an in vitro study using MC3T3-E1 osteoblastic cells to examine the effects of tension force (TF) on the expression of MMPs and TIMPs, and activation of mitogen-activated protein kinase (MAPK) pathways.Design: Cells were incubated on flexible-bottomed culture plates and stimulated with or without cyclic TF for 24 hours. The expression of MMPs and TIMPs was examined at mRNA and protein levels by real-time RT-PCR and Western blotting, respectively. The phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and stress-activated protein kinases/c-jun N-terminal kinases (SAPK/JNK) were examined by Western blotting.Results: TF decreased the expression of MMP-1, -3, -13 and phosphorylated ERK1/2. In contrast, TF increased the expression of TIMP-2, -3 and phosphorylated SAPK/JNK. The expression of MMP-2, -14, TIMP-1, -4 and phosphorylated p38 MAPK was unaffected by TF. MMP-1, -3 and -13 expression decreased in cells treated with the ERK inhibitor PD98059 compared with untreated control cells. The JNK inhibitor SP600125 inhibited the TF-induced upregulation of TIMP-2 and -3.Conclusions: The results suggest that TF suppresses the degradation process that occurs during ECM turnover in osteoid via decreased production of MMP-1, -3 and -13, and increased production of TIMP-2 and -3 through the MAPK signaling pathways in osteoblasts.     

Regulation of matrix metalloproteinases (MMPs) and their tissue inhibitors in human myometrial smooth muscle cells by TGF-beta1.

The objective of the present study was to determine whether transforming growth factor beta (TGF-beta) regulates the expression of matrix metalloproteinases (MMP) and the tissue inhibitor of MMP (TIMP) in myometrial smooth muscle cells. Using primary cultures of human myometrial smooth muscle cells we found that these cells express MMP-1, MMP-3, TIMP-1 and TIMP-2 mRNA and protein, with significantly higher values of TIMP than MMP. We also found that TGF-beta1 (1 ng/ml) increased the expression of TIMP-1 mRNA, while it reduced the expression of MMP-1 and MMP-3 mRNA, compared with untreated controls. In addition, TGF-beta1 slightly increased the production of TIMP-1, but not TIMP-2. Production of MMP-1 and MMP-3 was reduced by treatment with TGF-beta1, compared with the untreated control. A major portion of MMP-1 released into the culture-conditioned media was in complex with TIMP-1, and the levels of this complex were reduced by treatment with TGF-beta1. In conclusion, the data indicate that myometrial smooth muscle cells express MMP and TIMP mRNA and protein, and their expression is differentially regulated by TGF-beta1. Such a differential regulation of MMP and TIMP by TGF-beta may influence the rate of extracellular matrix (ECM) turnover following tissue injury, induced during myomectomy and Caesarean section, or in leiomyomas during growth.     

Matrix metalloproteinases and their inhibitors in the foreign body reaction on biomaterials

Matrix metalloproteinases (MMPs) can degrade structural components within the extracellular matrix and at the cellular surface producing changes in cellular behavior (i.e., adhesion and migration) and subsequent pathological responses (i.e., the foreign body reaction and wound healing). We continue to study the foreign body reaction that occurs following biomaterial implantation by investigating secretory responses of biomaterial-adherent macrophages and foreign body giant cells (FBGCs) as directed by material surface chemistry and further this research by determining whether secreted MMPs play a role in macrophage adhesion and fusion. We have identified numerous MMPs and their tissue inhibitors (TIMPs) in in vitro cell-culture supernatants using antibody arrays and quantified select MMP/TIMPs with ELISAs. MMP-9 concentrations were significantly greater than both TIMP-1 and TIMP-2 on all materials. The ratios of MMP-9/TIMP-1 and MMP-9/TIMP-2 increased with time because of an increase in MMP-9 concentrations over time, while the TIMP concentrations remained constant. Total MMP-9 concentrations in the supernatants were comparable on all materials at each timepoint, while TIMP-1 and TIMP-2 concentrations tended to be greater on hydrophilic/anionic surfaces. Analysis of the MMP/TIMP quantities produced per cell revealed that the hydrophilic/neutral surfaces, which inhibited macrophage adhesion, activated the adherent macrophages/FBGCs to produce a greater quantity of MMP-9, TIMP-1, and TIMP-2 per cell. Pharmacological inhibition of MMP-1,-8,-13, and -18 reduced macrophage fusion without affecting adhesion, while inhibitors of MMP-2,-3,-9, and -12 did not affect adhesion or fusion. These findings demonstrate that material surface chemistry does modulate macrophage/FBGC-derived MMP/TIMP secretion and implicates MMP involvement in macrophage fusion.     

Differential expression of matrix metalloproteinases and their inhibitors in non-small cell lung cancer

In a comprehensive immunohistochemical study of the expression of ten metalloproteinases (MMPs) and their four inhibitors (TIMPs) in 115 non-small cell lung carcinomas (NSCLCs), the findings have been correlated with the histological and clinical features of the tumours. All MMPs and TIMPs were expressed in tumours, with frequencies ranging from 41% for MMP-2 to 68% for MMP-13. Stromal immunoreactivity ranged from 6% for TIMP-4 to 87% for MMP-13. In some tumours, an overexpression of these proteins, as revealed by stronger staining in cancer cells than in adjacent normal bronchial epithelium, was also observed. The frequency ranged from 1% for MMP-3 to 28% for MMP-13. Compared with squamous cell carcinoma (SqCC), adenocarcinoma (AdC) more frequently overexpressed MMP-1, -11, -13, -14, and TIMP-2, and TIMP-1 and/or TIMP-2 overexpression positively correlated with more advanced stage disease. None of the MMP or TIMP expression correlated with the ras genotype of the tumours. The higher frequency of MMP overexpression in AdC than in SqCC may relate to the greater tendency of the former for systemic metastasis. The association of TIMP-1 overexpression with more advanced disease may suggest a role in prognosis.     

Role of metalloproteinases and tissue inhibitors of metalloproteinases during cardiopulmonary bypass in rats

Matrix metalloproteinases (MMPs) and the tissue inhibitors of matrix metalloproteinases (TIMPs) regulate matrix remodeling in the heart. Changes in synthesis and release of MMPs and TIMPs are observed after extracorporeal circulation (ECC). Thus, MMPs and TIMPs are supposed to be involved in ECC-mediated cardiac dysfunction. The aim was to examine the role of MMPs and TIMPs in ECC-mediated cardiac dysfunction. Extracorporeal circulation was instituted in rats for 60 min at a flow rate of 120 ml/kg/min. Three groups (n = 10) were studied: group CAO: 60 min ECC without aortic cross-clamping, group CAC: 60 min ECC including 30 min aortic cross-clamping (crystalloid Inzolen(?) cardioplegia), and group CAB: 60 min ECC including 30 min aortic cross-clamping (blood cardioplegia). Left ventricular (LV) function was measured with conductance catheter. Matrix metalloproteinase-activity was determined by zymography and TIMP activity was determined by reverse zymography. Gene expression of MMPs and TIMPs was determined by real-time polymerase chain reaction. Sixty minutes after weaning from bypass, there was a preserved LV function in the CAO and CAB group and an impaired LV function in the CAC group. We observed an increased myocardial activity and an increased myocardial messenger RNA expression of MMP-2, MMP-9, TIMP-1, and TIMP-4 in all ECC groups, when compared with sham animals. With regard to enzyme activity, there was an imbalance of MMP/TIMP ratio leading to an increased activity of MMP in the CAC group. In terms of gene expression, there was an imbalance of MMP-2/TIMP-4 ratio leading to an increased expression of MMP-2 in the CAC group. MMP-2 contributes to myocardial reperfusion injury in this in vivo model of ECC with cardioplegic arrest.     

Matrix metalloproteinase expression is related to angiogenesis and histologic grade in spindle cell soft tissue neoplasms of the extremities

We defined the immunocytochemical expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in benign soft tissue neoplasms, fibromatoses, and sarcomas, together with the activity of gelatinase MMPs and TIMPs measured by zymography and reverse zymography in a subset of cases. The most strongly expressed MMP in all tumors was MMP-1, with weaker expression of MMP-10, MMP-11, and MMP-14 in most tumors. Nuclear expression of MMP-1, MMP-8, and MMP-13 was an unusual feature. TIMP-2 was expressed in all tumors, with stronger expression in fibromatoses than in sarcomas. Fibromatoses and high-grade sarcomas showed greater MMP-1 expression than other groups, and endothelial MMP-2 expression was more extensive in sarcomas. Differences in MMP and TIMP expression might be linked to the biologic behavior of soft tissue neoplasms. The activation of endothelial MMP-2 linked to widespread MMP-14 expression provides a mechanism for sarcomas to modulate their matrix and facilitate angiogenesis.     

Regulation of matrix metalloproteinases and their inhibitors in the left ventricular myocardium of patients with aortic stenosis

Aortic stenosis (AS) results in myocyte and extracellular matrix remodeling in the human left ventricle (LV). The myocardial renin-angiotensin system is activated and collagens I and III and fibronectin accumulate. We determined the yet unknown regulation of enzymes that control collagen turnover, i.e., LV matrix metalloproteinases (MMP) and their tissue inhibitors (TIMPs) in human AS. We compared LV samples from AS patients undergoing elective aortic valve replacement (n=19) with nonused donor hearts with normal LV function (controls, n=12). MMP-2, MMP-9, MT1-MMP, and extracellular matrix metalloproteinase inducer (EMMPRIN), TIMP-1, TIMP-2, TIMP-3, and TIMP-4 mRNA were quantitated by real-time RCR. MMP-1, MMP-2, MMP-3, TIMP-3, TIMP-4, and EMMPRIN protein were measured by immunoblotting and MMP-9 and TIMP-1 protein by ELISA. Gelatinolytic MMP-2 and MMP-9 activity was measured by zymography. MMP-2 was increased in AS at mRNA, protein, and activity levels (131%, 193%, and 138% of controls). MMP-3 protein (308%) and EMMPRIN mRNA and protein were also upregulated (171% and 200%). In contrast, MMP-1 (37%) and MMP-9 mRNA, protein, and activity (26%, 21%, and 52%) were downregulated. MMP-9 activity was inversely correlated with LV size. TIMP-1 mRNA and protein were decreased (55% and 73%). In contrast, TIMP-2 mRNA (358%), TIMP-3 mRNA and protein (145% and 249%) were increased. TIMP-4 mRNA was not altered, but TIMP-4 protein was upregulated to 350%. Changes were similar in AS patients with normal and impaired LV ejection fraction. The dysregulation of myocardial MMPs and TIMPs in human AS starts at an early disease stage when LV function is still normal. In spite of upregulation of some MMPs the balance between MMP and TIMP is shifted towards MMP inhibition in human AS and may contribute to collagen accumulation.     

Platelet-derived growth factor and transforming growth factor-β modulate the expression of matrix metalloproteinases and migratory function of human airway smooth muscle cells

Background Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) have been suggested to be involved in the pathogenesis of asthma. Their expression in airway smooth muscle (ASM) cells could be involved in collagen turnover and migration of these cells and thus may contribute to airway remodelling.
Objective To examine the effect of pro-fibrotic growth factors TGF-β and platelet-derived growth factor (PDGF) on the expression of MMPs/TIMPs in cultured human ASM cells and to examine the role of MMP in the migration of ASM cells.
Methods ASM cells were stimulated with TGF-β and/or PDGF. Expression and activity of MMP-1, MMP-2, MMP-3, TIMP-1 and TIMP-2 were evaluated by quantitative RT-PCR, Western blot and zymography. Modified Boyden-chamber migration assay was performed to investigate the effect of secreted MMP-3 and TIMP-1 on ASM-cell migration.
Results PDGF strongly up-regulated the expression of MMP-1 at mRNA and protein levels. PDGF, when combined with TGF-β, caused synergistic up-regulation of MMP-3. TIMP-1 was additively up-regulated by TGF-β and PDGF. These growth factors had no effect on the expression of MMP-2 and TIMP-2. U0126, an extracellular signal-regulated kinase (ERK) pathway inhibitor, inhibited the up-regulation of MMP-1 by PDGF. The synergistic/additive up-regulation of MMP-3 and TIMP-1 was inhibited by U0126 and SB431542, a Smad pathway inhibitor. Supernatant from ASM cells in which MMP-3 production was knocked down by RNA interference showed a decreased migratory effect on ASM cells, whereas supernatant from cells with suppressed TIMP-1 expression resulted in increased migration.
Conclusion Our results suggest that PDGF with/without TGF-β could facilitate migration of ASM cells by modification of MMP–TIMP balance through the ERK pathway.     

Expression and Roles of MMP-2, MMP-9, MMP-13, TIMP-1, and TIMP-2 in Allergic Nasal Mucosa

Purpose

Allergic rhinitis (AR) and asthma share many characteristics, but structural changes are observed far less often in AR. Matrix metalloproteinases (MMPs) constitute a family of Zn-dependent endopeptidases that can decompose the extracellular matrix and basement membrane, and regulate cell infiltration. We analyzed the expression of MMPs and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), in allergic nasal mucosa after nasal allergen challenge (NAC) and determined their relationship to inflammatory cells.

Methods

Nasal mucosa specimens were obtained at surgery performed for hypertrophied turbinates. We performed NAC with house dust mite (HDM) allergen disks and control disks, and took biopsies at 30 minutes, 6 hours, and 12 hours after NAC. Cells expressing MMP-2, MMP-9, MMP-13, TIMP-1, and TIMP-2, as well as eosinophils and mast cells, were analyzed immunohistochemically. The MMPs and TIMPs in allergic nasal mucosa were quantified using enzyme-linked immunosorbent assays.

Results

At 30 minutes post-NAC, HDM-exposed nasal mucosa exhibited significantly more MMP-2+, MMP-9+, MMP-13+, TIMP-1+, and TIMP-2+ cells compared with control mucosa, and the numbers of MMP-9+ and TIMP-1+ cells correlated strongly with the number of mast cells. At 6 hours post-NAC, the numbers of MMP+ and TIMP+ cells did not differ significantly between HDM-exposed mucosa and control mucosa, but the ratios of MMP+ cells to TIMP+ cells were higher in HDM-exposed mucosa. At 12 hours post-NAC, the number of MMP-13+ cells tended to be higher in HDM-exposed mucosa and was strongly correlated with the number of eosinophils. Quantitatively, the levels of MMP-2 and MMP-13 were significantly higher than the MMP-9 level, and the TIMP-2 level was significantly higher than the TIMP-1 level in allergic nasal mucosa.

Conclusions

We demonstrated increased expression of MMP-2, MMP-9, and MMP-13 in allergic nasal mucosa, high MMPs-to-TIMP-1 ratios, and a strong correlation between MMP-9 and mast cells and between MMP-13 and eosinophils. The imbalance between MMPs and TIMPs may contribute to the migration of inflammatory cells such as eosinophils and mast cells to the nasal mucosa of AR patients, suggesting a possible active role of MMPs in AR.     

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases during normal human pulmonary development

AIMS : Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are thought to be involved in lung development because they play an important role in the turnover of the extracellular matrix. Although limited data on MMP and TIMP expression are available from animal studies during prenatal pulmonary development, little is known about their expression during human fetal lung development. The aim of this study was to investigate the expression of MMP-1, -2, -9, TIMP-1, -2 and -3 in human fetal lungs from 9 to 42 weeks of gestation. METHODS AND RESULTS : Forty-five normal human fetal lung samples were analysed by immunohistochemistry. MMP-1, -9, TIMP-1, -2 and -3, but not MMP-2, were expressed in the epithelium at all gestational ages. The endothelium of all vessels and the arterial smooth muscle cells expressed MMP-1, -2, -9, TIMP-2 and -3, but not TIMP-1, at all developmental stages. CONCLUSION : The extensive distribution of MMPs and TIMPs throughout all stages of human lung development suggests that they play a significant role in the remodelling that occurs in the interstitium and epithelial basement membrane during lung development and in pulmonary vascular development. These data will serve as a base line for comparison with neonatal lung pathology, including pulmonary hypertension.