Tissue Inhibitor Of Metalloproteinases-2 Research Articles

TIMP3/Wnt axis regulates gliosis of Müller glia

BackgroundPrevious studies have confirmed the expression of tissue inhibitor of metalloproteinase-3 (TIMP3) in Müller glia (MG). However, the role of TIMP3 in MG remains unknown. MethodsA mouse model of laser-induced retinal damage and gliosis was generated using wild-type C57BL/6 mice. TIMP3 and associated proteins were detected using Western blotting and immunofluorescence microscopy. RNA sequencing (GSE132140) of mouse laser-induced gliosis was utilized for pathway analysis. TIMP3 overexpression was induced in human MG. Human vitreous samples were obtained from patients with proliferative diabetic retinopathy (PDR) and healthy controls for protein analysis. ResultsTIMP3 levels increased in mouse eyes after laser damage. Morphology and spatial location of TIMP3 indicated its presence in MG. TIMP3-overexpressing MG showed increased cellular proliferation, migration, and cell nuclei size, suggesting TIMP3-induced gliosis for retinal repair. Glial fibrillary acidic protein (GFAP) and vimentin levels were elevated in TIMP3-overexpressing MG and laser-damaged mouse retinas. RNA sequencing and Western blotting suggested a role for β-catenin in mediating TIMP3 effects on the retina. Human vitreous samples from patients with PDR showed a positive correlation between TIMP3 and GFAP levels, both of which were elevated in patients with PDR. ConclusionsTIMP3 is associated with MG gliosis to enhance the repair ability of damaged retinas and is mediated by the canonical Wnt/β-catenin. Changes in TIMP3 could potentially be used to control gliosis in a range of retinal diseases However, given the multifaceted nature of TIMP3, care must be taken when developing treatments that aim solely to boost the function of TIMP3. FundingNational Cheng Kung University Hospital, Taiwan (NCKUH-10604009 and NCKUH-11202007); the Ministry of Science and Technology (MOST 110-2314-B-006-086-MY3).

The Role of Rosavin in the Pathophysiology of Bone Metabolism.

Rosavin, a phenylpropanoid in Rhodiola rosea's rhizome, and an adaptogen, is known for enhancing the body's response to environmental stress. It significantly affects cellular metabolism in health and many diseases, particularly influencing bone tissue metabolism. In vitro, rosavin inhibits osteoclastogenesis, disrupts F-actin ring formation, and reduces the expression of osteoclastogenesis-related genes such as cathepsin K, calcitonin receptor (CTR), tumor necrosis factor receptor-associated factor 6 (TRAF6), tartrate-resistant acid phosphatase (TRAP), and matrix metallopeptidase 9 (MMP-9). It also impedes the nuclear factor of activated T-cell cytoplasmic 1 (NFATc1), c-Fos, the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and mitogen-activated protein kinase (MAPK) signaling pathways and blocks phosphorylation processes crucial for bone resorption. Moreover, rosavin promotes osteogenesis and osteoblast differentiation and increases mouse runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) expression. In vivo studies show its effectiveness in enhancing bone mineral density (BMD) in postmenopausal osteoporosis (PMOP) mice, restraining osteoclast maturation, and increasing the active osteoblast percentage in bone tissue. It modulates mRNA expressions by increasing eukaryotic translation elongation factor 2 (EEF2) and decreasing histone deacetylase 1 (HDAC1), thereby activating osteoprotective epigenetic mechanisms, and alters many serum markers, including decreasing cross-linked C-telopeptide of type I collagen (CTX-1), tartrate-resistant acid phosphatase 5b (TRACP5b), receptor activator for nuclear factor κ B ligand (RANKL), macrophage-colony-stimulating factor (M-CSF), and TRAP, while increasing alkaline phosphatase (ALP) and OCN. Additionally, when combined with zinc and probiotics, it reduces pro-osteoporotic matrix metallopeptidase 3 (MMP-3), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α), and enhances anti-osteoporotic interleukin 10 (IL-10) and tissue inhibitor of metalloproteinase 3 (TIMP3) expressions. This paper aims to systematically review rosavin's impact on bone tissue metabolism, exploring its potential in osteoporosis prevention and treatment, and suggesting future research directions.

TIMP3 overexpression in myeloid lineage alleviates pancreatic damage and confers resistance to the development of type 1 diabetes in the MLDS -induced model.

Type 1 diabetes mellitus (T1DM) development involves a complex interplay of genetic, environmental, and immunological factors. By modulating the activity of proteases and receptors, the protein tissue inhibitor of metalloproteinase 3 (TIMP3) plays a role in limiting the expression and function of pro-inflammatory cytokines, which have been implicated in the advancement of T1DM. This study was aimed at examining the effect of TIMP3 overexpression in myeloid cells on the development of T1DM. Twelve weeks after multiple low doses of streptozotocin (MLDS) treatment, diabetic mice overexpressing TIMP3 specifically in myeloid cells under the CD68 promoter (MacT3 mice) showed improved insulin secretion, islet morphology and vascularization, antioxidant defense system, and regulatory factors of mitochondrial biosynthesis and function. To get mechanistic insights into the origin of this protection, the severity of insulitis and inflammatory parameters were evaluated in pancreatic tissues 11 days after MLSD treatment, showing significantly reduced insulitis and levels of the pro-inflammatory cytokine tumor necrosis factor-α, interleukin -1β, and interferon -γ in MacT3 mice. The results indicate that TIMP3 is involved in maintaining islet architecture and functions, at least in part, through modulation of pro-inflammatory cytokine production associated with insulitis and may represent a novel therapeutic strategy for T1DM.

Cellular behavior and extracellular matrix turnover in bovine annulus fibrosus cells under hydrostatic pressure and deviatoric strain.

Intervertebral disc herniation is a common spinal disorder that is often treated with discectomy when conservative measures fail. To devise therapeutic strategies for tears in the annulus fibrosus (AF), the regenerative capability of AF cells under spinal loading needs to be addressed. We hypothesized that the compressive loading associated with deformation in AF cells reduces synthetic and degradative activities in extracellular matrix and cell proliferation. We evaluated expression of key matrix molecules and cell proliferation by RT-PCR and immunohistochemistry by inner and outer bovine AF cells incubated under hydrostatic pressure (HP), arc-bending strain (Strain), and combined HP and Strain (HP/Strain) mimicking spinal loading. Inner AF cells showed significantly increased levels of aggrecan core protein, chondroitin sulfate N-acetylgalactosaminyltransferase-1, and tissue inhibitor of metalloproteinases-2 by 6 days under HP (p < 0.05), with a tendency toward increased matrix metalloproteinase-13. Outer AF cells demonstrated a significant decline in collagen type-2 under Strain and HP/Strain (p < 0.05) and a tendency toward suppression of collagen type-1 and elastin expression compared to HP and unloaded control. On the other hand, proliferating cell nucleus antigen increased significantly under Strain and HP/Strain in inner AF and declined under unloaded and HP in outer AF (p < 0.05). Immunohistology findings supported reductions in gene expressions of matrix molecules. Thus, changes in HP/Strain in AF appear to diminish synthetic and degradative activities while increasing cell proliferation. To promote regeneration, continuous overloading should be avoided, as it converts the synthetic activity to a state in which tissue repair is limited.

Sirt1 activation prevents high glucose-induced angiotensin converting enzyme 2 downregulation in renal tubular cells by regulating the TIMP3/ADAM17 pathway.

Angiotensin converting enzyme 2 (ACE2) exerts renoprotective effects in diabetic kidney disease (DKD) by converting angiotensin (Ang) II into Ang(1-7). Previous studies have demonstrated that ACE2 expression in renal tubules is downregulated in DKD, but the mechanism is not fully understood. Sirtuin-1 (Sirt1) is a protein deacetylase that may regulate the activity of the renin-angiotensin system. The present study investigated the effects of Sirt1 on ACE2 expression under high glucose (HG) conditions and the underlying signaling pathway. Rats with DKD and NRK-52E cells cultured with HG were employed in this study. Western blotting, immunohistochemistry detection and qRT-PCR were performed for protein and mRNA expression analyses. Rats subjected to DKD displayed downregulated expression of Sirt1 and ACE2 in kidneys. Resveratrol, an activator of Sirt1, restored ACE2 expression and ameliorated renal injuries. Similarly, pharmacological activation of Sirt1 with SRT1720 markedly upregulated ACE2 in NRK-52E cells cultured with HG, while Sirt1 small interfering RNA (siRNA) further suppressed ACE2 expression. In addition, A disintegrin and metalloproteinase (ADAM) 17 was observed to be upregulated, and its inhibitor, tissue inhibitor of metalloproteinase 3 (TIMP3), was downregulated in the kidneys of diabetic rats and NRK-52E cells incubated with HG. The TIMP3/ADAM17 pathway was involved in the regulation of ACE2 expression, as evidenced by decreased ACE2 expression levels after TIMP3-siRNA pretreatment. SRT1720 ameliorated the imbalance of TIMP3/ADAM17 induced by HG and consequently enhanced the expression of ACE2. Notably, the above effect of SRT1720 on ACE2 was interrupted by TIMP3-siRNA. Our findings suggest that Sirt1 activation may prevent HG-induced downregulation of renal tubular ACE2 by modulating the TIMP3/ADAM17 pathway. Sirt1 stimulation might be a potential strategy for the treatment of DKD.

Alterations in immunolocalisation of non-collagenous proteins in cartilage and bone tissue after gastrectomy, antrectomy and fundectomy in rat model

Bone, as a tissue, plays a pivotal role in maintaining whole body homeostasis, contributing to, among other things, structural support, blood cell production, and mineral storage. On the other hand, bone homeostasis depends on hormonal factors released by various tissues of the body, including the gastrointestinal tract. Thus, surgical procedures involving the removal of various parts of the stomach wall, such as, antrectomy and fundectomy or partial gastrectomy, may have profound negative effects on bone metabolism and mineralization. These effects may result from changes in the concentration of neuropeptides, such as nesfatin-1, secreted by cells located in the fundus and vestibule of the stomach. Additionally, bone morphogenetic proteins, osteocalcin, osteoprotegerin, and tissue inhibitor of metalloproteinases 2, are regulatory proteins involved in bone metabolism, playing a key role in maintaining the balance between the activity of osteoclasts and osteoblasts, cells responsible for the remodeling of bone tissue. The aim of this study was to investigate the effects of antrectomy, fundectomy, and partial gastrectomy, on bone homeostasis in a rat model. The experiment was carried out on 24 male Wistar rats randomly and equally divided into control (SHO), gastrectomy (GAST), antrectomy (ANT), and fundectomy (FUN) groups. At the beginning of the study, the rats underwent surgical removal of selected parts of the stomach. At the end of the 6-week study, the rats were sacrificed, and femurs were collected. Microscopic images of immunohistochemical reactions (IR) in trabecular and compact bone, as well as isolated articular cartilages and growth plates of femurs were analyzed. The intensity of the immunoreaction (IR) of osteocalcin (OC), osteoprotegerin (OPG), tissue inhibitor of metalloproteinases 2 (TIMP-2), bone morphogenetic protein 2 (BMP-2), and nesfatin-1 was determined, and IR-positive cells were counted in each area of the femur. The quantitative analysis of the intensity of OC IR in growth plate cartilage and trabecular bone showed the highest values after fundectomy in comparison to the other treatments. Moreover, fundectomy increased the intensity of BMP-2 IR in articular cartilage, growth plate cartilage, and trabecular bone. The intensity of OPG IR in articular cartilage and compact bone showed the highest values after antrectomy. The intensity of TIMP-2 IR in the examined parts of the femur was highest in the rats subjected to antrectomy. The intensity of expression of nesfatin-1 IR was highest in articular and growth plate cartilages for rats subjected to gastrectomy. In conclusion, surgical procedures affecting the gastrointestinal tract, such as antrectomy, fundectomy, and partial gastrectomy, can have significant effects on bone metabolism. Our study on a rat model demonstrated varying impacts on bone homeostasis, with different surgeries leading to alterations in the expression of key regulatory proteins involved in bone remodeling.

NDRG2 alleviates photoreceptor apoptosis by regulating the STAT3/TIMP3/MMP pathway in mice with retinal degenerative disease.

Photoreceptor apoptosis is the main pathological feature of retinal degenerative diseases; however, the underlying molecular mechanism has not been elucidated. Recent studies have shown that N-myc downstream regulated gene 2 (NDRG2) exerts a neuroprotective effect on the brain and spinal cord. In addition, our previous studies have confirmed that NDRG2 is expressed in mouse retinal photoreceptors and counteracts N-methyl-N-nitrosourea (MNU)-induced apoptosis. However, the underlying molecular mechanism remains unclear. In this study, we observed that the expression of NDRG2 was not only significantly inhibited in photoreceptors after MNU treatment but also after hydrogen peroxide treatment, and photoreceptor apoptosis was alleviated or aggravated after overexpression or knockdown of NDRG2 in the 661W photoreceptor cell line, respectively. The apoptosis inhibitor Z-VAD-FMK rescued photoreceptor apoptosis induced by MNU after NDRG2 knockdown. Next, we screened and identified tissue inhibitor of metalloproteinases 3 (TIMP3) as the downstream molecule of NDRG2 in 661W cells by using quantitative real-time polymerase chain reaction. TIMP3 exerts a neuroprotective effect by inhibiting the expression of matrix metalloproteinases (MMPs). Subsequently, we found that signal transducer and activator of transcription 3 (STAT3) mediated the NDRG2-associated regulation of TIMP3. Finally, we overexpressed NDRG2 in mouse retinal tissues by intravitreally injecting an adeno-associated virus with mouse NDRG2 in vivo. Results showed that NDRG2 upregulated the expression of phospho-STAT3 (p-STAT3) and TIMP3, while suppressing MNU-induced photoreceptor apoptosis and MMP expression. Our findings revealed how NDRG2 regulates the STAT3/TIMP3/MMP pathway and uncovered the molecular mechanism underlying its neuroprotective effect on mouse retinal photoreceptors.

Proteomic insights into the associations between obesity, lifestyle factors, and coronary artery disease

BackgroundWe aimed to investigate the protein pathways linking obesity and lifestyle factors to coronary artery disease (CAD).MethodsSummary-level genome-wide association statistics of CAD were obtained from the CARDIoGRAMplusC4D consortium (60,801 cases and 123,504 controls) and the FinnGen study (R8, 39,036 cases and 303,463 controls). Proteome-wide Mendelian randomization (MR) analysis was conducted to identify CAD-associated blood proteins, supplemented by colocalization analysis to minimize potential bias caused by linkage disequilibrium. Two-sample MR analyses were performed to assess the associations of genetically predicted four obesity measures and 13 lifestyle factors with CAD risk and CAD-associated proteins’ levels. A two-step network MR analysis was conducted to explore the mediating effects of proteins in the associations between these modifiable factors and CAD.ResultsGenetically predicted levels of 41 circulating proteins were associated with CAD, and 17 of them were supported by medium to high colocalization evidence. PTK7 (protein tyrosine kinase-7), RGMB (repulsive guidance molecule BMP co-receptor B), TAGLN2 (transgelin-2), TIMP3 (tissue inhibitor of metalloproteinases 3), and VIM (vimentin) were identified as promising therapeutic targets. Several proteins were found to mediate the associations between some modifiable factors and CAD, with PCSK9, C1S, AGER (advanced glycosylation end product-specific receptor), and MST1 (mammalian Ste20-like kinase 1) exhibiting highest frequency among the mediating networks.ConclusionsThis study suggests pathways explaining the associations of obesity and lifestyle factors with CAD from alterations in blood protein levels. These insights may be used to prioritize therapeutic intervention for further study.

CAR-T cells inhibit vascular smooth muscle phenotypic switching and reduce intimal hyperplasia in vein grafts

Abstract Background Intimal hyperplasia (IH) is a prevalent cause of vein graft stenosis after coronary artery bypass grafting (CABG) in the middle and long term. Vascular smooth muscle cell (VSMC) phenotypic switching from a contractile phenotype to a synthetic phenotype is a hallmark of this process, along with the degradation of extracellular matrix (ECM) and smooth muscle migration. Chimeric antigen receptor (CAR)-T cell therapy is a targeted cellular immunotherapy that uses genetically engineered T cells to specifically eliminate the antigen-bearing cells. There is a potential to apply CAR-T therapy to cardiovascular diseases. Purpose In this study, we aim to design a novel CAR-T cell therapy to eliminate synthetic vascular smooth muscle cells in intimal hyperplasia, inhibit phenotypic switching, in order to reduce vein graft stenosis. Methods and results Using bulk RNA-seq dataset from the GEO database, we have identified human organic cation transporter (OCT1) as a key marker for synthetic vascular smooth muscle cell in vein grafts (Fig.1A, B). We then engineered switchable fourth-generation CAR-T cells incorporating CD28 and CD3 ζ costimulatory domains (Fig.1C, E). In addition, these CAR-T cells secrete tissue inhibitor of metalloproteinase-3 (TIMP3), a protein that had been shown to inhibit matrix metalloproteinases (MMPs) activity and the ERK1/2 pathway (Fig. 1F). Targeting FITC instead of OCT1 itself allows a controllable and switchable working process for the CAR-T cells, as they only start to identify synthetic VSMCs in combination with a FITC-conjugated OCT1 antibody (Fig.1D). Immunostaining of PDGF-induced synthetic VSMCs co-cultured with CAR-T cells and FITC-conjugated OCT1 antibody revealed increased expression of contractile markers, and reduced expression of synthetic markers (Fig1.G), along with decreased mitochondrial membrane potentials, which could be reverted after pre-treatment with a pan caspase inhibitor (Fig.1H),. In vivo studies using mice vein graft model showed reduced intimal thickness, increased intimal/medial ratio in cross-sections, in addition to increased peak velocity and inner wall diameter on ultrasound after CAR-T cell therapy (Fig.2). Conclusion Switchable CAR-T cell therapy targeting synthetic vascular smooth muscle cells might be a promising approach to alleviate intimal hyperplasia in vein graft stenosis.

5 Rejuvenating Blood Factor TIMP2 Relates to Physical Activity and Cognitive Functioning in Older Adults on The Alzheimer’s Disease Continuum

Objective:Tissue inhibitor of metalloproteinases 2 (TIMP2) is produced peripherally, crosses the blood-brain barrier, and improves synaptic plasticity and hippocampal-dependent cognition in aged mice; however, the role of TIMP2 in human cognitive aging is unclear. We examined associations of circulating TIMP2 levels in blood with a known plasticity-inducing behavior, physical activity, and cognitive functioning among older adults along the Alzheimer’s disease continuum.Participants and Methods:Participants included 84 community-dwelling older adults (meanage = 78.8; 57% female; 82% cognitively normal; 14% MCI; 4% mild dementia; 35% PET Aß+) enrolled in the UC San Francisco Memory and Aging Center. All participants completed 30 days of observational FitbitTM monitoring to quantify physical activity (average daily steps), as well as a comprehensive in-person visit including blood draw (proteins assayed on SOMAscan platform), [18F]AV-45 positron emission tomography (PET) to quantify brain beta-amyloid (centiloids), and neuropsychological assessment. Composite cognitive z-scores were calculated for memory (California Verbal Learning Test-II [CVLT-II] and Benson Figure Recall), semantic processing (animal fluency and Boston Naming Test), and executive functioning (digits backwards span, Stroop inhibition, modified trail making test, lexical fluency, and design fluency). Multiple linear regression examined TIMP2 as a function of physical activity, covarying for age and PET centiloids. Additional regression models separately examined cognitive z-scores as a function of TIMP2, covarying for age, sex, education, PET centiloids, and body mass index (BMI).Results:TIMP2 was not significantly correlated with age, sex, education, or PET centiloids (ps &gt; 0.05); however, TIMP2 was negatively correlated with BMI (r = -0.23, p = 0.036). Greater average daily steps related to higher levels of TIMP2 (b = 0.30, 95%CI = 0.04-0.55, p = 0.022). TIMP2 also related to better semantic processing (b = 0.28, 95%CI = 0.04-0.51, p = 0.021) and executive functioning (b = 0.26, 95%CI = 0.03-0.49, p = 0.028). TIMP2 did not significantly relate to memory (p &gt; 0.05).Conclusions:Greater physical activity was associated with higher concentrations of blood factor TIMP2, which in turn related to better cognitive functioning independent of Alzheimer’s disease pathology burden. These results support previous mouse models by broadly replicating relationships between TIMP2 and cognition in humans, while also uniquely demonstrating an association between TIMP2 and physical activity, a modifiable protective factor in both typical and diseased cognitive aging. Our domain-specific results, however, suggest that benefits of TIMP2 in humans may involve a broader neuroanatomical network than the hippocampal-specific effects previously shown in mice. Although exact mechanisms of TIMP2 need further examination, TIMP2 is known to be enriched in human umbilical cord plasma, has been shown to be involved in cell-growth promoting activities, and may relate to increased neural plasticity in older age. Further examination of TIMP2 and other novel blood-based proteins as potential therapeutic targets for improved cognitive aging, including in the presence of Alzheimer’s disease, is warranted.

Targeting multiple disease hallmarks using a synergistic disease-modifying drug combination ameliorates osteoarthritis via inhibition of senescence and inflammation

AimsOsteoarthritis (OA), is a debilitating disease characterized by progressive cartilage degradation, synovial inflammation, and chondrocyte senescence. Various treatment agents independently targeting these hallmarks have been investigated. However, due to the complex multifaceted nature of OA, no disease-modifying osteoarthritis drugs are clinically available. In an attempt to overcome this, we developed a combinatorial approach and demonstrated the efficacy of TsC [Tissue inhibitor of metalloproteinase-3 (TIMP3) + sulfated carboxymethylcellulose (sCMC)] and piperlongumine (PL) combination for the amelioration of OA in a goat ex vivo OA model. Main methodsThe efficacy of the drug combination was evaluated using the goat ex vivo OA explant model and results were validated in clinically relevant human OA cartilage explants. The chondroprotective effects were evaluated in terms of reduced inflammation and cartilage matrix loss, reduction in chondrosenescence, and reduced oxidative stress. Key findingsA combination of TsC and PL (TsC-PL) significantly reduced inflammation, cartilage matrix loss, chondrosenescence, and oxidative stress in the goat ex vivo OA model and showed chondroprotective effects. Further, similar chondroprotective effects were observed in human OA cartilage. Additionally, the coefficient of drug interaction analysis indicated that the combination of TsC and PL had a synergistic effect in reducing matrix degrading proteases and inflammation (goat ex vivo OA model) and Reactive oxygen species (ROS) production (human OA cartilage). SignificanceCombinatorial treatment with TsC and PL demonstrated potential disease-modifying effects for the treatment of osteoarthritis via inhibition of inflammation and senescence and supports the usage of treatment strategies targeting multiple pathological factors of OA simultaneously.

Neuronal TIMP2 regulates hippocampus-dependent plasticity and extracellular matrix complexity

Functional output of the hippocampus, a brain region subserving memory function, depends on highly orchestrated cellular and molecular processes that regulate synaptic plasticity throughout life. The structural requirements of such plasticity and molecular events involved in this regulation are poorly understood. Specific molecules, including tissue inhibitor of metalloproteinases-2 (TIMP2) have been implicated in plasticity processes in the hippocampus, a role that decreases with brain aging as expression is lost. Here, we report that TIMP2 is highly expressed by neurons within the hippocampus and its loss drives changes in cellular programs related to adult neurogenesis and dendritic spine turnover with corresponding impairments in hippocampus-dependent memory. Consistent with the accumulation of extracellular matrix (ECM) in the hippocampus we observe with aging, we find that TIMP2 acts to reduce accumulation of ECM around synapses in the hippocampus. Moreover, its deletion results in hindrance of newborn neuron migration through a denser ECM network. A novel conditional TIMP2 knockout (KO) model reveals that neuronal TIMP2 regulates adult neurogenesis, accumulation of ECM, and ultimately hippocampus-dependent memory. Our results define a mechanism whereby hippocampus-dependent function is regulated by TIMP2 and its interactions with the ECM to regulate diverse processes associated with synaptic plasticity.

Dznep, a histone modification inhibitor, inhibits HIF1α binding to TIMP2 gene and suppresses TIMP2 expression under hypoxia.

Epidemiological studies have shown that patients who recovered from acute kidney injury (AKI) may subsequently develop chronic kidney disease (CKD). AKI is primarily caused by renal hypoxia, and it causes epigenetic alterations, known as hypoxic memory. 3-Deazaneplanocin A (Dznep), an inhibitor of histone modification, suppresses renal fibrosis and the expression of tissue inhibitor of metalloproteinases-2 (TIMP2), a profibrotic factor, in mouse ischemia-reperfusion models. The current study investigated the epigenetic regulation of TIMP2 in human kidney 2 (HK-2) cells. The expression of TIMP2 was upregulated in HK-2 cells under hypoxic conditions and was suppressed by Dznep. ChIP-qPCR showed that Dznep reduced the amount of H3K4me3 at the promoter region of the TIMP2 gene under hypoxic condition. Formaldehyde-assisted isolation of regulatory elements-qPCR of the TIMP2 gene showed that Dznep reduced open chromatin area. In addition, based on ChIP-qPCR of hypoxia-inducible factor 1 alpha (HIF1α), Dznep inhibited the binding of HIF1α to the TIMP2 gene under hypoxic conditions. The reporter assays for the binding region of HIF1α showed enhanced transcriptional activity by hypoxia. Dznep suppresses the expression of TIMP2 under hypoxic conditions by inhibiting the binding of HIF1α to the TIMP2 gene.

The relationship between atherogenic indices and tissue inhibitor of metalloproteinase-3 in chronic heart failure

The relationship between atherogenic indices and tissue inhibitor of metalloproteinase-3 in chronic heart failure

Suramin analogues protect cartilage against osteoarthritic breakdown by increasing levels of tissue inhibitor of metalloproteinases 3 (TIMP-3) in the tissue

Osteoarthritis is a chronic degenerative joint disease affecting millions of people worldwide, with no disease-modifying drugs currently available to treat the disease. Tissue inhibitor of metalloproteinases 3 (TIMP-3) is a potential therapeutic target in osteoarthritis because of its ability to inhibit the catabolic metalloproteinases that drive joint damage by degrading the cartilage extracellular matrix. We previously found that suramin inhibits cartilage degradation through its ability to block endocytosis and intracellular degradation of TIMP-3 by low-density lipoprotein receptor-related protein 1 (LRP1), and analysis of commercially available suramin analogues indicated the importance of the 1,3,5-trisulfonic acid substitutions on the terminal naphthalene rings for this activity. Here we describe synthesis and structure–activity relationship analysis of additional suramin analogues using ex vivo models of TIMP-3 trafficking and cartilage degradation. This showed that 1,3,6-trisulfonic acid substitution of the terminal naphthalene rings was also effective, and that the protective activity of suramin analogues depended on the presence of a rigid phenyl-containing central region, with para/para substitution of these phenyl rings being most favourable. Truncated analogues lost protective activity. The physicochemical characteristics of suramin and its analogues indicate that approaches such as intra-articular injection would be required to develop them for therapeutic use.

Targeting of HBP1/TIMP3 axis as a novel strategy against breast cancer

Malignant proliferation and metastasis are the main causes of breast cancer death. The transcription factor high mobility group (HMG) box-containing protein 1 (HBP1) is an important tumor suppressor whose deletion or mutation is closely related to the appearance of tumors. Here, we investigated the role of HBP1 in breast cancer suppression. HBP1 enhances the activity of the tissue inhibitors of metalloproteinases 3 (TIMP3) promoter, thereby increasing protein and mRNA levels of TIMP3. TIMP3 increases the phosphatase and tensin homolog (PTEN) protein level by inhibiting its degradation and acts as a metalloproteinase inhibitor to inhibit the protein levels of MMP2/9. In this study, we demonstrated that the HBP1/TIMP3 axis plays a crucial role in inhibiting the tumorigenesis of breast cancer. HBP1 deletion interferes with the regulation of the axis and induces the occurrence and malignant progression of breast cancer. In addition, the HBP1/TIMP3 axis promotes the sensitivity of breast cancer to radiation therapy and hormone therapy. Our study opens new perspectives on the treatment and prognosis of breast cancer.

#5471 THE URINARY BIOMARKERS IGFBP7 AND IGFBP7 X TIMP2 PRE-EMPTIVELY IDENTIFY PATIENTS AT RISK OF CONTRAST NEPHROTOXICITY

Abstract Background and Aims Drug nephrotoxicity is a serious medical and economic concern. In fact, 25% of the 100 most used drugs in intensive care units are toxic to the kidney, which limits their use and, therefore, the correct therapy for the patient. The administration of contrast media (CM) during diagnostic tests or surgical interventions carries the risk of contrast-induced nephropathy (CIN), which is a clinical condition defined as an increase in plasma creatinine of 25% or 0.5 mg/dL above baseline within 3-5 days after administration. Because once installed CIN has no treatment, identification of new biomarkers predicting patients at risk of CIN before receiving CM may be crucial to prevent future kidney complications. Clinical studies conducted in other medical areas have identified new urinary biomarkers, such as insulin like growth factor binding protein 7 (IGFBP7) and tissue inhibitor of metalloproteinases 2 (TIMP2), that anticipate the risk of developing kidney damage after the administration of different potentially nephrotoxic compounds. The objective of this work was to evaluate the usefulness of these biomarkers to predict CIN in cardiac patients. Method A clinical study was carried out with 154 patients from the University Hospital of Salamanca Cardiology Department who subsequently received a CM. Prior to contrast administration, urine samples were collected. In addition, their plasma creatinine level was registered for the following 5 days to evaluate the development of CIN. Patients were divided into Controls (who did not develop CIN) and Cases (who developed CIN). IGFBP7 and TIMP2 biomarkers were quantified in urine samples by ELISA. Subsequently, the differences between both groups were evaluated using the Mann-Whitney U test and the diagnostic capacity of each biomarker was analyzed through the generation of its receiver operating characteristic (ROC) curve. Results Of the total number of patients, 123 were assigned to the Controls group and 31 to the Cases group. In both groups the distribution of sex and risk factors was similar, except for the case of age and body mass index, which was slightly lower and higher, respectively, in the Controls group. IGFBP7 was significantly higher in Cases (p &amp;lt; 0.01) compared to the Controls, and the IGFBP7 x TIMP2 product further improved this significance (p &amp;lt; 0.001). Specifically, the area under the ROC curve for IGFBP7 was 0.67 (95% confidence interval of 0.56-0.78); while that for IGFBP7 x TIMP2 increased to 0.73 (with a 95% confidence interval of 0.63-0.84). In contrast, TIMP2 alone showed no differences between both groups of patients. Conclusion The biomarkers IGFBP7 and IGFBP7 x TIMP2 could therefore be used for the prophylactic identification and management of patients at risk of developing CIN.

Urinary Kidney Injury Biomarkers Are Associated with Ischemia-Reperfusion Injury Severity in Kidney Allograft Recipients.

We explored the potential of emerging and conventional urinary kidney injury biomarkers in recipients of living donor (LD) or donation after circulatory death (DCD) kidney transplantation, patients with chronic kidney disease (CKD), and individuals from the general population. Urine samples from kidney allograft recipients with mild (LD; n = 199) or severe (DCD; n = 71) ischemia-reperfusion injury (IRI) were analyzed for neutrophil gelatinase-associated lipocalin (NGAL), insulin-like growth factor-binding protein 7 (IGFBP7), tissue inhibitor of metalloproteinases 2 (TIMP2), kidney injury molecule-1 (KIM-1), chemokine C-X-C motif (CXCL9), solute carrier family 22 member 2 (SLC22A2), nephrin, and uromodulin (UMOD) by quantitative multiplex LC-MS/MS analysis. The fold-change in biomarker levels was determined in mild and severe IRI and in patients with CKD stage 1-2 (n = 127) or stage ≥3 (n = 132) in comparison to the general population (n = 1438). Relationships between the biomarkers and total protein, β2-microglobulin (B2M), creatinine, and osmolality were assessed. NGAL, IGFBP7, TIMP2, KIM-1, CXCL9, and UMOD were quantifiable, whereas nephrin and SLC22A2 were below the limit of detection. Kidney injury biomarkers were increased up to 6.2-fold in allograft recipients with mild IRI and 8.3-fold in recipients with severe IRI, compared to the reference population, with the strongest response observed for NGAL and B2M. In CKD stage 1-2, B2M, NGAL, IGFBP7, TIMP2, KIM-1, UMOD, and CXCL9 were not altered, but in individuals with CKD stage ≥3, B2M, NGAL, and KIM-1 were increased up to 1.3-fold. IGFBP7, TIMP2, NGAL, and CXCL9 were strongly correlated (all r ≥ 0.8); correlations with B2M and TP were smaller (all r ≤ 0.6). IRI, but not stable CKD, was associated with increased urinary levels of kidney injury biomarkers determined by LC-MS/MS. Absolute and multiplexed protein quantitation by LC-MS/MS is an effective strategy for biomarker panel evaluation for translation toward the clinical laboratory.

Noncanonical Activity of Tissue Inhibitor of Metalloproteinases 2 (TIMP2) Improves Cognition and Synapse Density in Aging.

Peripheral administration of tissue inhibitor of metalloproteinases 2 (TIMP2), a protein inhibitor of matrix metalloproteinases (MMPs), has previously been shown to have beneficial effects on cognition and neurons in aged mice. Here, to better understand the potential of recombinant TIMP2 proteins, an IgG4Fc fusion protein (TIMP2-hIgG4) was developed to extend the plasma half-life of TIMP2. Following one month of administration of TIMP2 or TIMP2-hIgG4 via intraperitoneal injections, 23-month-old male C57BL/6J mice showed improved hippocampal-dependent memory in a Y-maze, increased hippocampal cfos gene expression, and increased excitatory synapse density in the CA1 and dentate gyrus (DG) of the hippocampus. Thus, fusion to hIgG4 extended the half-life of TIMP2 while retaining the beneficial cognitive and neuronal effects. Moreover, it retained its ability to cross the blood-brain barrier. To deepen the mechanistic understanding of the beneficial function of TIMP2 on neuronal activity and cognition, a TIMP2 construct lacking MMP inhibitory activity, Ala-TIMP2, was generated, which provides steric hindrance that prevents inhibition of MMPs by the TIMP2 protein while still allowing MMP binding. A comprehensive assessment of the MMP inhibitory and binding capacity of these engineered proteins is outlined. Surprisingly, MMP inhibition by TIMP2 was not essential for its beneficial effects on cognition and neuronal function. These findings both confirm previously published research, expand on the potential mechanism for the beneficial effects of TIMP2, and provide important details for a therapeutic path forward for TIMP2 recombinant proteins in aging-related cognitive decline.

PhosY-secretome profiling combined with kinase-substrate interaction screening defines active c-Src-driven extracellular signaling

c-Src tyrosine kinase is a renowned key intracellular signaling molecule and a potential target for cancer therapy. Secreted c-Src is a recent observation, but how it contributes to extracellular phosphorylation remains elusive. Using a series of domain deletion mutants, we show that the N-proximal region of c-Src is essential for its secretion. The tissue inhibitor of metalloproteinases 2 (TIMP2) is an extracellular substrate of c-Src. Limited proteolysis-coupled mass spectrometry and mutagenesis studies verify that the Src homology 3 (SH3) domain of c-Src and the P31VHP34 motif of TIMP2 are critical for their interaction. Comparative phosphoproteomic analyses identify an enrichment of PxxP motifs in phosY-containing secretomes from c-Src-expressing cells with cancer-promoting roles. Inhibition of extracellular c-Src using custom SH3-targeting antibodies disrupt kinase-substrate complexes and inhibit cancer cell proliferation. These findings point toward an intricate role for c-Src in generating phosphosecretomes, which will likely influence cell-cell communication, particularly in c-Src-overexpressing cancers.