Human Tissue Inhibitor Of Metalloproteinases Research Articles

High-affinity ssDNA aptamer and chemiluminescent aptasensor for TIMP-1 detection in human serum

TIMP-1 (Tissue Inhibitor of Metalloproteinases-1) is a protein involved in regulating extracellular matrix (ECM) degradation. It is recognized as a significant biomarker for cancer diagnosis. This study aimed to develop and characterize a single-stranded DNA (ssDNA) aptamer targeting human TIMP-1 protein with high affinity and specificity. A magnetic beads-based SELEX process combined with qPCR was used to select aptamers over seven rounds. The enriched ssDNA library was analyzed using high-throughput sequencing to identify candidate sequences, and these sequences were characterized using surface plasmon resonance (SPR) and binding assays to evaluate their affinity and specificity. The selected ssDNA aptamer demonstrated a dissociation equilibrium constant (KD) of 0.41 nM and a very slow off-rate, enabling effective capture of TIMP-1 in serum samples. Furthermore, a chemiluminescent aptasensor was developed for TIMP-1 detection, which exhibited high specificity and a broad linear detection range from 1 to 500 ng/mL in human serum. The developed ssDNA aptamer targeting TIMP-1 shows high affinity and specificity, and the chemiluminescent aptasensor demonstrates promising potential for clinical diagnosis of TIMP-1 levels in human serum.Graphical abstract

Engineering minimal tissue inhibitors of metalloproteinase targeting MMPs via gene shuffling and yeast surface display.

Overexpression of specific matrix metalloproteinases (MMPs) has a key role in development of several diseases, such as cancer, neurological disorders, and cardiovascular diseases due to their critical role in degradation and remodeling of the extracellular matrix (ECM). Tissue inhibitors of metalloproteinases (TIMPs), a family of four in humans, are endogenous inhibitors of MMPs. TIMPs have a high level of sequence and structure homology, with a broad range of binding and inhibition to the family of MMPs. It is important to identify the key motifs of TIMPs responsible for inhibition of MMPs to developefficient therapeutics targeting specific MMPs. We used DNA shuffling between the human TIMP family to generate a minimal TIMP hybrid library in yeast to identify the dominant minimal MMP inhibitory regions. The minimal TIMP variants screened toward MMP-3 and MMP-9 using fluorescent-activated cell sorting (FACS). Interestingly, several minimal TIMP variants selected after screening toward MMP-3cd or MMP-9cd, with lengths as short as 20 amino acids, maintained or improved binding to MMP-3 and MMP-9. The TIMP-MMP binding dissociation constant (KD ), in the nM range, and MMP inhibition constants (Ki ), in the pM range, of these minimal TIMP variants were similar to the N-terminal domain of TIMP-1 on the yeast surface and in solution indicating the potency of these minimal variants as MMP inhibitors. We further used molecular modeling simulation, and molecular docking of the minimal TIMP variants in complex with MMP-3cd to understand the binding and inhibition mechanism of these variants.

Human TIMP1 Is a Growth Factor That Improves Oocyte Developmental Competence.

Oocyte developmental competence is the ability of a mature oocyte to be fertilized and subsequently support embryonic development. Such competence is gained during folliculogenesis and is facilitated by the bidirectional communication into a compacted cumulus-oocyte complex (COC). Human tissue inhibitor of metalloproteinases-1 (TIMP1) participates in biological processes, including cell growth, differentiation, and apoptosis. This study aimed to evaluate the influence of TIMP1 as a growth factor on the in vitro maturation (IVM) culture of bovine COCs to improve oocyte developmental competence. All TIMP1 treatments (50, 100, and 150 ng/mL) favored the COCs' compaction structure (p < 0.05). TIMP1 at 150 ng/mL produced more oocytes in metaphase II compared to the other treatments (p < 0.05). The 150 ng/mL TIMP1 generated oocytes with the most (p < 0.05) cortical granules below the plasma membrane (pattern I). In a parthenogenesis assay, oocyte IVM in 50 ng/mL of TIMP1 produced the most blastocyst compared to the other treatments (p < 0.05). The Principal Component Analysis (PCA) showed that 50 ng/mL of TIMP1 was the best condition to develop oocyte competence because it was associated with the COC compact and cortical granule pattern I. TIMP1 influences the development of oocyte competence when added to the IVM culture medium of COCs.

Identification of a Tissue Inhibitor of Metalloproteinase-2: An Endogenous Inhibitor of Modori-Inducing Insoluble Metalloproteinase from Yellowtail Seriola quinqueradiata Muscle.

The existence of an endogenous protease inhibitor (EPI) was expected from the comparison of the gel properties between washed and nonwashed yellowtail surimi gels. A possible candidate, tissue inhibitor of metalloproteinase-2 (TIMP-2), was partially purified from the soluble fraction of yellowtail muscle, and an 18 kDa protein band was detected by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions and western blot analysis. Its N-terminal amino acid sequence was determined as XSXSPAHPQQAF, with high homology to TIMP-2 from other fish species, suggesting that it was identified as yellowtail TIMP-2. Subsequently, full-length cDNA of two isoforms (TIMP-2a and TIMP-2b) was successfully cloned from yellowtail muscle. The N-terminal sequence of purified TIMP-2 completely corresponded to TIMP-2b. When the surimi gel quality decreased after spawning, the mRNA expression of TIMP-2b also decreased. Human TIMP-2 could inhibit autolysis of myofibrillar proteins from yellowtail muscle. Thus, TIMP-2b was considered the major EPI of the modori-inducing insoluble metalloproteinase in yellowtail muscle.

Relationship Between Endothelial and Angiogenesis Biomarkers Envisage Mortality in a Prospective Cohort of COVID-19 Patients Requiring Respiratory Support.

PurposeEndothelial damage and angiogenesis are fundamental elements of neovascularisation and fibrosis observed in patients with coronavirus disease 2019 (COVID-19). Here, we aimed to evaluate whether early endothelial and angiogenic biomarkers detection predicts mortality and major cardiovascular events in patients with COVID-19 requiring respiratory support.MethodsChanges in serum syndecan-1, thrombomodulin, and angiogenic factor concentrations were analysed during the first 24 h and 10 days after COVID-19 hospitalisation in patients with high-flow nasal oxygen or mechanical ventilation. Also, we performed an exploratory evaluation of the endothelial migration process induced by COVID-19 in the patients' serum using an endothelial cell culture model.ResultsIn 43 patients, mean syndecan-1 concentration was 40.96 ± 106.9 ng/mL with a 33.9% increase (49.96 ± 58.1 ng/mL) at day 10. Both increases were significant compared to healthy controls (Kruskal–Wallis p < 0.0001). We observed an increase in thrombomodulin, Angiopoietin-2, human vascular endothelial growth factor (VEGF), and human hepatocyte growth factor (HGF) concentrations during the first 24 h, with a decrease in human tissue inhibitor of metalloproteinases-2 (TIMP-2) that remained after 10 days. An increase in human Interleukin-8 (IL-8) on the 10th day accompanied by high HGF was also noted. The incidence of myocardial injury and pulmonary thromboembolism was 55.8 and 20%, respectively. The incidence of in-hospital deaths was 16.3%. Biomarkers showed differences in severity of COVID-19. Syndecan-1, human platelet-derived growth factor (PDGF), VEGF, and Ang-2 predicted mortality. A multiple logistic regression model with TIMP-2 and PDGF had positive and negative predictive powers of 80.9 and 70%, respectively, for mortality. None of the biomarkers predicted myocardial injury or pulmonary thromboembolism. A proteome profiler array found changes in concentration in a large number of biomarkers of angiogenesis and chemoattractants. Finally, the serum samples from COVID-19 patients increased cell migration compared to that from healthy individuals.ConclusionWe observed that early endothelial and angiogenic biomarkers predicted mortality in patients with COVID-19. Chemoattractants from patients with COVID-19 increase the migration of endothelial cells. Trials are needed for confirmation, as this poses a therapeutic target for SARS-CoV-2.

Features of the level of matrix metalloproteinase-2, -3, -9 and tissue inhibitors of metalloproteinases-1, -2, -3, -4 in the aqueous humor of patients with primary open-angle glaucoma

Aim. To study the content of matrix metalloproteinase (MMP)-2, -3, -9 and tissue inhibitors of metalloproteinases (TIMPs) -1, -2, -3, -4 in the aqueous humor of patients with moderate primary open-angle glaucoma (POAG).Materials and methods. The experimental group included 47 patients with verified moderate primary open-angle glaucoma. The control group consisted of 26 patients with uncomplicated cataract. The levels of MMP-2, -3, -9 were determined with Luminex Performance Human MMP Magnetic Panel 3-plex kit (R&amp;D Systems, USA), the concentration of TIMPs-1, -2, -3, - 4 was determined with the Human TIMP Magnetic Luminex Performance Assay 4-plex kit (R&amp;D Systems, USA). The study was carried out using flow-through field fluorometry on a Bio-Plex 200 double-beam laser analyzer (Bio-Rad, USA).Results. The study showed a statistically significant increase in the levels of matrix metalloproteinase-2 and tissue inhibitors of matrix metalloproteinases-1, -2, -3, -4 in the aqueous humor of patients with moderate POAG compared with patients with uncomplicated cataract.Conclusion. The obtained data on high concentrations and imbalance in the levels of matrix metalloproteinases and their tissue inhibitors in the aqueous humor of patients with moderate POAG confirm the role of local inflammation, as well as impairments in the structure of the extracellular matrix and its remodeling in the mechanisms of development of this pathology.

ISSLS prize in basic science 2021: a novel inducible system to regulate transgene expression of TIMP1

PurposeInflammatory and oxidative stress upregulates matrix metalloproteinase (MMP) activity, leading to intervertebral disc degeneration (IDD). Gene therapy using human tissue inhibitor of metalloproteinase 1 (hTIMP1) has effectively treated IDD in animal models. However, persistent unregulated transgene expression may have negative side effects. We developed a recombinant adeno-associated viral (AAV) gene vector, AAV-NFκB-hTIMP1, that only expresses the hTIMP1 transgene under conditions of stress.MethodsRabbit disc cells were transfected or transduced with AAV-CMV-hTIMP1, which constitutively expresses hTIMP1, or AAV-NFκB-hTIMP1. Disc cells were selectively treated with IL-1β. NFκB activation was verified by nuclear translocation. hTIMP1 mRNA and protein expression were measured by RT-PCR and ELISA, respectively. MMP activity was measured by following cleavage of a fluorogenic substrate.ResultsIL-1β stimulation activated NFκB demonstrating that IL-1β was a surrogate for inflammatory stress. Stimulating AAV-NFκB-hTIMP1 cells with IL-1β increased hTIMP1 expression compared to unstimulated cells. AAV-CMV-hTIMP1 cells demonstrated high levels of hTIMP1 expression regardless of IL-1β stimulation. hTIMP1 expression was comparable between IL-1β stimulated AAV-NFκB-hTIMP1 cells and AAV-CMV-hTIMP1 cells. MMP activity was decreased in AAV-NFκB-hTIMP1 cells compared to baseline levels or cells exposed to IL-1β.ConclusionAAV-NFκB-hTIMP1 is a novel inducible transgene delivery system. NFκB regulatory elements ensure that hTIMP1 expression occurs only with inflammation, which is central to IDD development. Unlike previous inducible systems, the AAV-NFκB-hTIMP1 construct is dependent on endogenous factors, which minimizes potential side effects caused by constitutive transgene overexpression. It also prevents the unnecessary production of transgene products in cells that do not require therapy.

Coupling of a Novel TIMP3 Peptide to Carboxypeptidase G2 for Pro-Drug Activation at the Tumour Site.

Broad-spectrum cytotoxic drugs have been used in cancer therapy for decades. However, their lack of specificity to cancer cells often results in serious side-effects, limiting efficacy. For this reason, antibodies have been used to attempt to specifically target cytotoxic drugs to tumours. One such approach is antibody-directed enzyme prodrug therapy (ADEPT) which uses a tumour-directed monoclonal antibody, coupled to an enzyme, to convert a systemically administered non-toxic prodrug into a toxic one only at the tumour site. Among the main drawbacks of ADEPT is the immunogenicity of the antibody-enzyme complex, which is exacerbated by slow clearance due to size, hence limiting repeated administration. Additionally, the mono-specificity of the antibody could potentially result in drug resistance with repeated administration. We have identified a novel short peptide sequence, p700, derived from a human tissue inhibitor of metalloproteinases-3 (TIMP-3), which binds to and inhibits a number of tyrosine kinase growth factor receptors (VEGFRs1-3, FGFRs 1-4 and PDGFRα) which are known to be upregulated in many tumours and tumour vasculature. In this report, we fused p700 to His-tagged, codon-optimised, carboxypeptidase G2 (CPG2). CPG2 is a bacterial enzyme used in ADEPT, which activates potent nitrogen-mustard pro-drugs by removal of an inhibitory glutamic acid residue. Recombinant CPG2-p700 was highly expressed in Escherichia coli and successfully purified by nickel affinity chromatography. Biolayer interferometry showed that CPG2-p700 had a 100-fold increase in binding affinity for VEGFR2 compared with CPG2 alone and retained its catalytic activity, as determined by methotrexate cleavage. In the presence of CPG2-p700, the ZD2676P pro-drug showed significant cytotoxicity for 4T1 cells compared with prodrug alone or CPG2 alone. p700 is, therefore, a potentially useful alternative to monoclonal antibodies for enzyme pro-drug therapy and could equally be used for effective delivery of other cytotoxic drugs to tumour tissue.

Prokaryotic expression, purification and characterization of tissue inhibitor of metalloproteinase-2

Tissue inhibitor of metalloproteinases-2 (TIMP-2) inhibits tumor migration and invasion. Obtaining TIMP-2 protein is conducive to a comprehensive and in-depth study of its function and mechanism in tumorigenesis and development. We collected human TIMP-2 protein through prokaryotic expression in vitro. We expressed, purified and characterized human TIMP-2 protein. First, the human TIMP-2 gene was cloned from the cDNA obtained by reverse transcription of total RNA of human lung cancer A549 cells, and constructed to pET28a vector. The recombinant plasmid pET28a-TIMP-2 was transformed into Escherichia coli BL21(DE3) after restriction endonuclease digestion and sequencing analysis. The expression of TIMP-2 protein was induced by isopropyl-β-D-thiogalactoside (IPTG), and the expression conditions were optimized. After purification by nickel affinity column, the fusion protein His-TIMP-2 was identified by Western blotting method and its biological activity was detected by gelatin zymography. The fusion protein His-TIMP-2 existed in the form of inclusion body in E. coli. In a certain range, the concentration of IPTG had no significant effect on the expression amount of His-TIMP-2. But in this expression system, induction temperature and time were the key parameters, and the expression amount of His-TIMP-2 in E. coli increased with the increase of induction temperature. The purified and refolded fusion protein could effectively inhibit the activity of matrix metalloproteinases expressed by human lung cancer A549 cells. The acquisition of active fusion protein lays a foundation for further study of the function and mechanism of human TIMP-2, and is of great significance for tumor therapy.

The expression, purification, and substrate analysis of matrix metalloproteinases in Drosophila melanogaster

Matrix metalloproteinases (MMPs) are evolutionarily conserved extracellular matrix proteinases. Genetic analysis of the Drosophila MMPs, Mmp1 and Mmp2, in vivo reveal that they play vital roles in tissue remodeling. Although the catalytic domain (CD) undertakes most MMP functions, few studies have sought to demonstrate the biochemical properties of the CDs of fly MMPs. Here, we identified the overexpression, purification, and refolding of the CDs of Drosophila Mmp1 and Mmp2 for biochemical studies. Zymography assays and substrate degradation analysis showed that both Mmp1-CD and Mmp2-CD were able to digest casein, gelatin, fibronectin, collagen (types I, IV, and V), while Mmp2-CD showed much higher degradation activity compared with Mmp1-CD. Moreover, human collagen III could be degraded by Mmp1-CD but not Mmp2-CD, and rat collagen I and laminin could be degraded by Mmp2-CD but not Mmp1-CD, suggesting that Drosophila Mmp1 and Mmp2 might have overlapping yet distinct substrate specificity. Using synthetic fluorescent substrates, we further demonstrated that the enzymatic activity of Mmp1-CD and Mmp2-CD could be inhibited by human tissue inhibitors of metalloproteinases (TIMPs). These results reveal the context of the cooperative yet distinct roles of Mmp1 and Mmp2 in tissue remodeling.

Human tissue inhibitor of metalloproteinases-1 improved wound healing in diabetes through its anti-apoptotic effect.

Impaired wound healing accompanies severe cell apoptosis in diabetic patients. Tissue inhibitor of metalloproteinases-1 (TIMP-1) was known to have effects on promoting growth and anti-apoptosis for cells. We aimed to determine the actual levels of TIMP-1 and cell apoptosis in: (i) the biopsies of diabetic and non-diabetic foot tissue and (ii) the human fibroblasts with or without treatments of advanced glycation end-products (AGEs). Next, we aimed to determine the improved levels of cell apoptosis and wound healing after the treatments of either active protein of TIMP-1 or in vivo expression of gene therapy vector-mediated TIMP-1 in both the human fibroblasts and the animal model of diabetic rats. The levels of TIMP-1 were significantly reduced in diabetic skin tissues and in AGEs-treated fibroblasts. Both AGEs-treated cells were effectively protected from apoptosis by active protein of TIMP-1 at appropriate dose level. So did the induced in vivo TIMP-1 expression after gene delivery. Similar effects were also found on the significant improvement of impaired wound healing in diabetic rats. We concluded that TIMP-1 improved wound healing through its anti-apoptotic effect. Treatments with either active protein TIMP-1 or TIMP-1 gene therapy delivered in local wound sites may be used as a strategy for accelerating diabetic wound healing.

Effects of overexpression of human tissue inhibitor of metalloproteinase-1 on the inflammatory response in rats with myocardial infarction and related mechanisms

Objective: To observe the effects of recombinant adenovirus with human tissue inhibitor of metalloproteinase-1(Ad-hTIMP-1) on the inflammatory response in rats with myocardial infarction (MI) and explore the related mechanisms. Methods: The male Wistar rats were randomly divided into sham-operated group, saline group, Ad-Track group and Ad-hTIMP-1 group according to the random number table (n=8 each group). MI was induced by ligation of the left anterior descending coronary artery and MI rats were injected with saline, Ad-Track and Ad-hTIMP-1, respectively. Sham-operated rats received similar surgical procedure without ligation of the left anterior descending coronary artery. After 4 weeks, the cardiac function was measured by echocardiography, then rats were sacrificed and hearts were removed for morphological and biological analysis. The morphology of myocardial tissue in each group was detected by HE staining and Masson staining. The mRNA expressions of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10 and C-reactive protein(CRP) were detected by real-time PCR. Immune histochemical staining was performed to observe the protein expression levels of IL-6 and CRP. Results: (1) Left ventricular end systolic dimension derived from echocardiography was increased in saline group ((5.10±0.72) mm) and Ad-Track group ((4.88±0.64) mm) compared to sham-operated group ((4.25±0.46) mm), which was reduced in Ad-hTIMP-1 group ((4.13±0.35) mm, all P<0.05). The left ventricular ejection fraction was (72.46±5.74)%, (64.27±8.52)%, (64.65±3.90)%, and (71.55±6.95)%, the fractional shortening was (36.90±4.97)%, (29.03±3.40)%, (30.95±2.51)%, and (36.31±5.68)% in sham-operated group, saline group, Ad-Track group and Ad-hTIMP-1 group, respectively. The left ventricular ejection fraction and fractional shortening in saline group and Ad-Track group were lower than those in sham-operated group and Ad-hTIMP-1 group (all P<0.05). (2) Necrosis of myocardial cells was not found and a small amount of immune cell infiltration and interstitial fibrosis were observed on HE and Masson stained myocardial sections of Ad-hTIMP-1 group. (3) Real-time PCR showed that mRNA expressions of TNF-α, IL-6, IL-10 and CRP were lower in Ad-hTIMP-1 group than in saline group. mRNA expressions of TNF-α, IL-10 and CRP were lower in Ad-hTIMP-1 group than in Ad-Track group (all P<0.05). (4) Immune histochemical staining showed that protein expressions of IL-6 and CRP were higher in saline group and Ad-Track group than those in Ad-hTIMP-1 group (all P<0.05). Conclusion: Recombinant adenovirus Ad-hTIMP-1 can improve cardiac function in rats with myocardial infarction via inhibiting the inflammatory response and downregulating the expression of TNF-α, IL-6 and CRP.

Direct expression of active human tissue inhibitors of metalloproteinases by periplasmic secretion in Escherichia coli

BackgroundAs regulators of multifunctional metalloproteinases including MMP, ADAM and ADAMTS families, tissue inhibitors of metalloproteinases (TIMPs) play a pivotal role in extracellular matrix remodeling, which is involved in a wide variety of physiological processes. Since abnormal metalloproteinase activities are related to numerous diseases such as arthritis, cancer, atherosclerosis, and neurological disorders, TIMPs and their engineered mutants hold therapeutic potential and thus have been extensively studied. Traditional productions of functional TIMPs and their N-terminal inhibitory domains (N-TIMPs) rely on costly and time-consuming insect and mammalian cell systems, or tedious and inefficient refolding from denatured inclusion bodies. The later process is also associated with heterogeneous products and batch-to-batch variation.ResultsIn this study, we developed a simple approach to directly produce high yields of active TIMPs in the periplasmic space of Escherichia coli without refolding. Facilitated by disulfide isomerase (DsbC) co-expression in protease-deficient strain BL21 (DE3), N-TIMP-1/-2 and TIMP-2 which contain multiple disulfide bonds were produced without unwanted truncations. 0.2–1.4 mg purified monomeric TIMPs were typically yielded per liter of culture media. Periplasmically produced TIMPs exhibited expected inhibition potencies towards MMP-1/2/7/14, and were functional in competitive ELISA to elucidate the binding epitopes of MMP specific antibodies. In addition, prepared N-TIMPs were fully active in a cellular context, i.e. regulating cancer cell morphology and migration in 2D and 3D bioassays.ConclusionPeriplasmic expression in E. coli is an excellent strategy to recombinantly produce active TIMPs and N-TIMPs.

Differential expression and localization of human tissue inhibitors of metalloproteinases in proliferative diabetic retinopathy.

Tissue inhibitors of metalloproteinases (TIMPs) block the catalysis by matrix metalloproteinases (MMPs) and have additional biologic activities, including regulation of cell growth and differentiation, apoptosis, angiogenesis and oncogenesis. We investigated the expression levels of all the four human TIMPs and correlated these levels with those of MMP-9 and vascular endothelial growth factor (VEGF) in proliferative diabetic retinopathy (PDR). Vitreous samples from 38 PDR and 21 nondiabetic control patients and epiretinal membranes from 14 patients with PDR and 10 patients with proliferative vitreoretinopathy (PVR) were studied by enzyme-linked immunosorbent assay, Western blot analysis and immunohistochemistry. Tissue inhibitor of metalloproteinases-1, TIMP-4, MMP-9 and VEGF levels were significantly higher in vitreous samples from PDR patients than in nondiabetic controls (p<0.0001 for all comparisons), whereas TIMP-2 and TIMP-3 levels did not differ significantly. TIMP-1, TIMP-4, MMP-9 and VEGF levels in PDR with active neovascularization were significantly higher than those in inactive PDR (p<0.0001, 0.001, 0.013, 0.004, respectively). Significant positive correlations existed between levels of TIMP-1 and levels of TIMP-4 (r=0.37; p=0.004), MMP-9 (r=0.65; p<0.0001) and VEGF (r=0.59; p<0.0001), between levels of TIMP-4 and levels of MMP-9 (r=0.61; p<0.0001) and VEGF (r=0.62; p<0.0001) and between levels of MMP-9 and VEGF (r=0.62; p<0.0001). TIMP-1 and TIMP-3 were expressed in vascular endothelial cells in PDR epiretinal membranes and in myofibroblasts and leucocytes in PDR and PVR epiretinal membranes. The differential expression of TIMPs in PDR suggests that among the 4 TIMPs, TIMP-1 and TIMP-4 may be possible biomarkers of disease activity.

The tissue inhibitor of metalloproteinases-1 (TIMP-1) promotes survival and migration of acute myeloid leukemia cells through CD63/PI3K/Akt/p21 signaling.

We and others have shown that the Tissue Inhibitor of Metalloproteinases-1 (TIMP-1), a member of the inflammatory network exerting pleiotropic effects in the bone marrow (BM) microenvironment, regulates the survival and proliferation of different cell types, including normal hematopoietic progenitor cells. Moreover, TIMP-1 has been shown to be involved in cancer progression. However, its role in leukemic microenvironment has not been addressed. Here, we investigated the activity of TIMP-1 on Acute Myelogenous Leukemia (AML) cell functions. First, we found that TIMP-1 levels were increased in the BM plasma of AML patients at diagnosis. In vitro, recombinant human (rh)TIMP-1 promoted the survival and cell cycle S-phase entry of AML cells. These kinetic effects were related to the downregulation of cyclin-dependent kinase inhibitor p21. rhTIMP-1 increases CXCL12-driven migration of leukemic cells through PI3K signaling. Interestingly, activation of CD63 receptor was required for TIMP-1's cytokine/chemokine activity. Of note, rhTIMP-1 stimulation modulated mRNA expression of Hypoxia Inducible Factor (HIF)-1α, downstream of PI3K/Akt activation. We then co-cultured AML cells with normal or leukemic mesenchymal stromal cells (MSCs) to investigate the interaction of TIMP-1 with cellular component(s) of BM microenvironment. Our results showed that the proliferation and migration of leukemic cells were greatly enhanced by rhTIMP-1 in presence of AML-MSCs as compared to normal MSCs. Thus, we demonstrated that TIMP-1 modulates leukemic blasts survival, migration and function via CD63/PI3K/Akt/p21 signaling. As a “bad actor” in a “bad soil”, we propose TIMP-1 as a potential novel therapeutic target in leukemic BM microenvironment.

Aldosterone Induces Tissue Inhibitor of Metalloproteinases-1 Expression and Further Contributes to Collagen Accumulation: From Clinical to Bench Studies.

Aldosterone induces myocardial fibrosis. Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a key factor of myocardial fibrosis. This study tested the hypothesis that aldosterone induces TIMP-1 expression and contributes to the fibrotic process. We prospectively enrolled 54 patients with primary aldosteronism, and measured plasma TIMP-1 and echocardiographic parameters. In the cell study, we investigated the possible molecular mechanism by which aldosterone induces TIMP-1 secretion and the effects on collagen accumulation. In the animal study, we measured serum TIMP-1 levels, cardiac TIMP-1 levels, and cardiac structure in an aldosterone infusion mouse model using implantation of aldosterone pellets. In patients with primary aldosteronism, plasma TIMP-1 was correlated with 24-hour urinary aldosterone, left ventricular mass, and impairment of left ventricular diastolic function. In human cardiac fibroblasts, TIMP-1 protein and mRNA expressions were significantly increased by aldosterone through the glucocorticoid receptor/PI3K/Akt/nuclear factor-κB pathway. TIMP-1 small-interfering RNA significantly reduced aldosterone-induced collagen accumulation, and aldosterone did not alter the levels of collagen1a1 or matrix metalloproteinase-1 mRNA. The aldosterone-induced TIMP-1 expression was inversely related to matrix metalloproteinase-1 activity. Furthermore, in the animal model, the serum and cardiac levels of TIMP-1 were significantly elevated in the mice that received aldosterone infusion. This elevation was blocked by RU-486 but not by eplerenone, suggesting that the effect was through glucocorticoid receptors. In a long-term aldosterone infusion model, serum TIMP-1 was associated with serum aldosterone level, cardiac structure, and fibrosis. In conclusion, aldosterone induced TIMP-1 expression in vivo and in vitro. This increased TIMP-1 expression resulted in enhanced collagen accumulation via the suppression of matrix metalloproteinase-1 activity.

The tissue inhibitor of metalloproteinases 1 increases the clonogenic efficiency of human hematopoietic progenitor cells through CD63/PI3K/Akt signaling

Initially described as an endogenous inhibitor of proteases, the tissue inhibitor of metalloproteinases 1 (TIMP-1) also displays cytokine-like functions. TIMP-1 is a soluble protein whose levels are increased under inflammatory conditions. We recently found that TIMP-1(-/-) mice have decreased bone marrow (BM) cellularity and that the engraftment capability of TIMP-1(-/-) hematopoietic stem cells (HSCs) is impaired, owing to proliferation defects. Here, we investigated the role of recombinant human TIMP-1 (rhTIMP-1) in human hematopoietic stem/progenitor cells (HSPCs) and elucidated the downstream pathway ignited by rhTIMP-1. We found that rhTIMP-1 affects invitro cell survival, proliferation, and particularly clonogenic expansion of CD34(+) HSPCs without compromising their short-term engraftment potential after transplantation into immunodeficient mice. These effects are independent on matrix metalloproteinase (MMP) inhibition and rely on TIMP-1's binding to the tetraspanin membrane receptor CD63. Further investigation indicated that rhTIMP-1 stimulation induces phosphatidylinositol 3-kinase (PI3K) recruitment and Akt phosphorylation, both presiding over survival/proliferation pathways in HSPCs. Downstream targets of phosphorylated Akt (pAkt) are also modulated, including the proliferation marker cyclin D1 (CycD1), whose levels are increased upon exposure to rhTIMP-1. These findings indicate that rhTIMP-1 promotes clonogenic expansion and survival in human progenitors via the activation of the CD63/PI3K/pAkt signaling pathway, suggesting that TIMP-1 might be a key player in the network of proinflammatory factors modulating HSPC functions.

Purification and characterization of bioactive his6-tagged recombinant human tissue inhibitor of metalloproteinases-1 (TIMP-1) protein expressed at high yields in mammalian cells

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is an endogenous inhibitor of matrix metalloproteinases (MMPs) with reported tumor promoting, as well as inhibitory, effects. These paradoxical properties are presumably mediated by different biological functions, MMP-dependent as well as -independent, and probably related to TIMP-1 levels of protein expression, post-translational modifications, and cellular localization. TIMP-1 is an N-glycosylated protein that folds into two functional domains, a C- and an N-terminal domain, with six disulfide bonds. Furthermore, TIMP-1 is processed in the N-terminal sequence. These three biochemical properties make TIMP-1 difficult to produce in conventional bacterial, insect, or yeast expression systems. We describe here a HEK293 cell-based strategy for production and purification of secreted and N-glycosylated recombinant his6-tagged human TIMP-1 (his6-rTIMP-1), which resulted in large amounts of highly purified and bioactive protein. Matrix-assisted laser desorption ionization mass spectrometry confirmed the N- and C-termini of his6-rTIMP-1, and N-glycosylation profiling showed a match to the N-glycosylation of human plasma TIMP-1. The his6-rTIMP-1 was bioactive as shown by its proper inhibitory effect on MMP-2 activity, and its stimulatory effect on cell growth when added to the growth medium of four different breast cancer cell lines. This study provides an easy set-up for large scale production and purification of bioactive, tagged recombinant human TIMP-1, which structurally and functionally is similar to endogenous human TIMP-1, while using an expression system that is adaptable to most biochemical and biomedical laboratories including those that do not perform protein purifications routinely.

Human Adipose-Derived Stromal/Stem Cells Protect Against STZ-Induced Hyperglycemia: Analysis of hASC-Derived Paracrine Effectors

Adipose-derived stromal/stem cells (ASCs) ameliorate hyperglycemia in rodent models of islet transplantation and autoimmune diabetes, yet the precise human ASC (hASC)-derived factors responsible for these effects remain largely unexplored. Here, we show that systemic administration of hASCs improved glucose tolerance, preserved β cell mass, and increased β cell proliferation in streptozotocin-treated nonobese diabetic/severe combined immunodeficient mice. Coculture experiments combining mouse or human islets with hASCs demonstrated that islet viability and function were improved by hASCs following prolonged culture or treatment with proinflammatory cytokines. Analysis of hASC-derived factors revealed vascular endothelial growth factor and tissue inhibitor of metalloproteinase 1 (TIMP-1) to be highly abundant factors secreted by hASCs. Notably, TIMP-1 secretion increased in the presence of islet stress from cytokine treatment, while TIMP-1 blockade was able to abrogate in vitro prosurvival effects of hASCs. Following systemic administration by tail vein injection, hASCs were detected in the pancreas and human TIMP-1 was increased in the serum of injected mice, while recombinant TIMP-1 increased viability in INS-1 cells treated with interleukin-1beta, interferon-gamma, and tumor necrosis factor alpha. In aggregate, our data support a model whereby factors secreted by hASCs, such as TIMP-1, are able to mitigate against β cell death in rodent and in vitro models of type 1 diabetes through a combination of local paracrine as well as systemic effects.

Effects of Transplantation of hTIMP-1–Expressing Bone Marrow Mesenchymal Stem Cells on the Extracellular Matrix of Degenerative Intervertebral Discs in an In Vivo Rabbit Model

Prospective, randomized, and controlled animal study. To observe extracellular matrix (ECM) changes in degenerative intervertebral disc (IVD) after transplantation of bone marrow mesenchymal stem cells (BMSCs) virally transfected with a construct expressing "human tissue inhibitor of metalloproteinase 1" (hTIMP-1), and to discuss the feasibility of using this approach to treat IVD degeneration. Intervertebral disc (IVD) degeneration is characterized by decreased cell numbers, bioactivity of the nucleus pulposus, and remodeled ECM. Exogenous genes can be targeted into cells to produce inhibition of ECM degradation and increase ECM content in IVDs, and thereby potentially stop or reverse degenerative processes and modify disc structure. BMSCs were isolated from a pure New Zealand white rabbit and identified by flow cytometry. Transgenic BMSCs were acquired by transfection with a recombinant adenovirus vector carrying the hTIMP-1 gene. Animal models of IVD degeneration were established by annulus puncture and then given intra-nucleus pulposus injections according to their random assignment into 3 groups: (1) a transgenic BMSC transplantation (TgBT) group that received BMSCs transfected with an hTIMP-1-expressing adenovirus vector; (2) a BMSC transplantation (BT) group that received unaltered BMSCs; and (3) a control group that received cell-free phosphate-buffered saline. Degree of degeneration was evaluated 12 weeks after modeling. ECM content was quantified using immunohistochemistry and spectrophotography. Expression of hTIMP-1 was observed via quantitative polymerase chain reaction, western blot, and immunohistochemistry. Significantly fewer degenerative changes and increased ECM content were observed in the TBT and BT groups than the control group animals (P < 0.05). The TBT group had greater ECM content than did the BT group (P < 0.05), as well as higher levels of hTIMP-1 mRNA and protein. Transplantation of BMSCs transfected with hTIMP-1 can increase ECM content by inhibiting ECM degradation and promoting ECM synthesis. N/A.