MicroRNA-197-3p mediates damage to human coronary artery endothelial cells via targeting TIMP3 in Kawasaki disease.

Kawasaki disease (KD) causes cardiovascular system injury in children. However, the pathogenic mechanisms of KD have not been well defined. Recently, strong correlation between aberrant microRNAs and KD nosogenesis has been revealed. A role of microRNA-197-3p (miR-197-3p) in the pathogenesis of KD is identified in the present study. Cell proliferation assay showed human coronary artery endothelial cells (HCAECs) were suppressed by serum from KD patients, which was correlated with high levels of miR-197-3p in both KD serum and HCAECs cultured with KD serum. The inhibition of HCAECs by miR-197-3p was confirmed by cells expressing miR-197-3p mimic and miR-197-3p inhibitor. Comparative proteomics analysis and Ingenuity Pathway Analysis (IPA) revealed TIMP3 as a potential target of miR-197-3p, which was demonstrated by western blot and dual-luciferase reporter assays. Subsequently, by detecting the endothelium damage markers THBS1, VWF, and HSPG2, the role of miR-197-3p/TIMP3 in KD-induced damage to HCAECs was confirmed, which was further validated by a KD mouse model in vivo. The expressions of miR-197-3p and its target, TIMP3, are dramatically variational in KD serum and HCAECs cultured with KD serum. Increased miR-197-3p induces HCAECs abnormal by restraining TIMP3 expression directly. Hence, dysregulation of miR-197-3p/TIMP3 expression in HCAECs may be an important mechanism in cardiovascular endothelium injury in KD patients, which offers a feasible therapeutic target for KD treatment.

Pioglitazone Attenuates Lupus Nephritis Symptoms in Mice by Modulating miR-21-5p/TIMP3 Axis: the Key Role of the Activation of Peroxisome Proliferator-Activated Receptor-γ.

Lupus nephritis (LN) is a severe symptom of systemic lupus erythematosus and miR-21-5p is upregulated during LN. In the current study, the effects of pioglitazone (Pg), a peroxisome proliferator-activated receptor-γ (PPARγ) agonist, on LN development were assessed and explained by focusing miR-21-5p/TIMP3 axis. The expressions of miR-21-5p and PPARγ in LN mice were detected and then the mice were treated with pioglitazone to evaluate the anti-LN effects of agent. The miR-21-5p level was induced in MRL/lpr mice to confirm the central role of miR-21-5p inhibition in the protective effects of Pg against LN. The level of miR-21-5p was upregulated, while the level of PPARγ was downregulated in MRL/lpr mice. Pg inhibited miR-21-5p in renal tissues, which induced the expression of TIMP3. The changes in miR-21-5p/TIMP3 axis led to the improvements in renal structure and function, and inhibited autoimmune response. The induction of miR-21-5p impaired the effects of Pg, along with the suppression of TIMP3. The expression of miR-21-5p was associated with the progression of LN, contributing to the suppression of TIMP3 and development of LN. The inhibition of the miR-21-5p by Pg would restore the structure and function of kidneys in LN mice via the activation of PPARγ.

TIMP-3 facilitates binding of target metalloproteinases to the endocytic receptor LRP-1 and promotes scavenging of MMP-1.

Matrix metalloproteinases (MMPs) and the related families of disintegrin metalloproteinases (ADAMs) and ADAMs with thrombospondin repeats (ADAMTSs) play a crucial role in extracellular matrix (ECM) turnover and shedding of cell-surface molecules. The proteolytic activity of metalloproteinases is post-translationally regulated by their endogenous inhibitors, known as tissue inhibitors of metalloproteinases (TIMPs). Several MMPs, ADAMTSs and TIMPs have been reported to be endocytosed by the low-density lipoprotein receptor-related protein-1 (LRP-1). Different binding affinities of these proteins for the endocytic receptor correlate with different turnover rates which, together with differences in their mRNA expression, determines their nett extracellular levels. In this study, we used surface plasmon resonance to evaluate the affinity between LRP-1 and a number of MMPs, ADAMs, ADAMTSs, TIMPs and metalloproteinase/TIMP complexes. This identified MMP-1 as a new LRP-1 ligand. Among the proteins analyzed, TIMP-3 bound to LRP-1 with highest affinity (KD = 1.68 nM). Additionally, we found that TIMP-3 can facilitate the clearance of its target metalloproteinases by bridging their binding to LRP-1. For example, the free form of MMP-1 was found to have a KD of 34.6 nM for LRP-1, while the MMP-1/TIMP-3 complex had a sevenfold higher affinity (KD = 4.96 nM) for the receptor. TIMP-3 similarly bridged binding of MMP-13 and MMP-14 to LRP-1. TIMP-1 and TIMP-2 were also found to increase the affinity of target metalloproteinases for LRP-1, albeit to a lesser extent. This suggests that LRP-1 scavenging of TIMP/metalloproteinase complexes may be a general mechanism by which inhibited metalloproteinases are removed from the extracellular environment.

Overexpression of TIMP3 inhibits discogenic pain by suppressing angiogenesis and the expression of substance P in nucleus pulposus.

Approximately 50% of the cases of low back pain (LBP) are attributed to discogenic origin. The causes of discogenic pain are complicated and consist of a complex biochemical cascade. Neovascularization of intervertebral discs (IVDs) is believed to be associated with discogenic pain. The anti­angiogenesis ability of tissue inhibitor of metalloproteinase­3 (TIMP3) has been reported in many tumors, yet whether TIMP3 is associated with neovascularization of IVDs remains unknown. In the present study, both in vitro and in vivo models were used to investigate the association between discogenic pain and TIMP3 expression in nucleus pulposus (NP). PCR results demonstrated that inflammation induced downregulation of TIMP3 expression in NP cells. By using an adenovirus system to upregulate TIMP3 expression, the effect of TIMP3 on angiogenesis was measured by endothelial cell migration and tube formation assays. The results demonstrated that overexpression of TIMP3 suppressed angiogenesis in NP without the regulation of vascular endothelial growth factor (VEGF) expression. TNF­α converting enzyme (TACE) expression was downregulated by TIMP3, thus inhibiting the TACE­induced activation of TNF­α in NP cells. Immunohistochemical staining of IVDs also confirmed that TIMP3 inhibited the expression of substance P in NP. Taken together, the present results indicated the expression of TIMP3 in NP may have a key role in the development of discogenic pain.

LRP1 Controls TNF Release via the TIMP-3/ADAM17 Axis in Endotoxin-Activated Macrophages.

The metalloproteinase ADAM17 plays a pivotal role in initiating inflammation by releasing TNF from its precursor. Prolonged TNF release causes many chronic inflammatory diseases, indicating that tight regulation of ADAM17 activity is essential for resolution of inflammation. In this study, we report that the endogenous ADAM17 inhibitor TIMP-3 inhibits ADAM17 activity only when it is bound to the cell surface and that cell surface levels of TIMP-3 in endotoxin-activated human macrophages are dynamically controlled by the endocytic receptor LRP1. Pharmacological blockade of LRP1 inhibited endocytic clearance of TIMP-3, leading to an increase in cell surface levels of the inhibitor that blocked TNF release. Following LPS stimulation, TIMP-3 levels on the surface of macrophages increased 4-fold within 4 h and continued to accumulate at 6 h, before a return to baseline levels at 8 h. This dynamic regulation of cell surface TIMP-3 levels was independent of changes in TIMP-3 mRNA levels, but correlated with shedding of LRP1. These results shed light on the basic mechanisms that maintain a regulated inflammatory response and ensure its timely resolution.

TIMP-3 suppression induces choroidal neovascularization by moderating the polarization of macrophages in age-related macular degeneration.

PURPOSE: To investigate the role of tissue inhibitor of metalloproteinases-3 (TIMP-3) as a key moderator of macrophage polarization in choroidal neovascularization (CNV) lesions of model mice and in bone marrow-derived macrophage (BMDM). METHOD: We used siR-TIMP-3 to transfect BMDM and gave an intravitreal injection of siR-TIMP-3 to laser-induced CNV mice model, real time-PCR and western blot were applied for detecting the expressions of TIMP-3 and macrophages' biomarker. Besides, CNV lesions in different treatment groups of animal model were examined by the optical coherence tomography angiography (OCTA). RESULTS: Our experimental data showed that lack of TIMP-3 stimulated M2 polarization proved by real time-PCR and western blot in BMDMs and CNV mice model. Moreover, intravitreal injection of siR-TIMP-3 accelerated CNV formation using OCTA, which indicated that TIMP-3 suppression is related to pro-angiogenesis of M2 macrophage. CONCLUSION: We showed that the absence of TIMP-3 leads to a more pro-angiogenic microenvironment, playing a key role in CNV formation by positively modulating M2 polarization. The role of TIMP-3 in the regulating inflammation and novel therapeutic target of nAMD needs to be further studied.

MicroRNA-323a-3p Promotes Pressure Overload-Induced Cardiac Fibrosis by Targeting TIMP3.

BACKGROUND/AIMS: Cardiac fibrosis is a major cause of diverse cardiovascular diseases. MicroRNAs have recently been proven a novel class of regulators of cardiac fibrosis. In this study, we sought to investigate the role of miR-323a-3p and its mechanisms in regulating cardiac fibrosis. METHODS: The transverse aortic constriction (TAC) mice model was induced and neonatal cardiac fibroblasts (CFs) were cultured. MTT (3- [4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay was used to detect the cell viability. Echocardiography was used to evaluate cardiac function. Masson's Trichrome stain was used to evaluate the development of fibrosis. Luciferase activity assay was performed to confirm the miRNA's binding site. Real-time PCR and Western blot were used to evaluate the level of mRNA and protein. RESULTS: MiR-323a-3p was found up-regulated in myocardial tissues subjected to TAC and in CFs cultured with Angiotensin Ⅱ (Ang Ⅱ). Overexpression of miR-323a-3p significantly increased the mRNA levels of collagen Ⅰ, collagen Ⅲ, MMP2 and MMP9, while inhibition of miR-323a-3p prevented the proliferation, collagen production and the protein level of transforming growth factor (TGF-ß) in rat neonatal CFs. Strikingly, injection of antagomiR-323a-3p elevated cardiac function and inhibited the expression of TGF-ß in the TAC mice. TIMP3 was a direct target of miR-323a-3p, as the overexpression of miR-323a-3p decreased the protein and mRNA levels of TIMP3. In the CFs with pre-treatment of Ang Ⅱ, siRNA-TIMP abolished the effects of AMO-323a-3p on the inhibition of the proliferation of CFs, the down-regulation of collagen Ⅰ and collagen Ⅲ, and the expression of TGF-ß. CONCLUSION: Our findings provide evidence that miR-323a-3p promotes cardiac fibrosis via miR-323a-3p-TIMP3-TGF-ß pathway. miR-323a-3p may be a new marker for cardiac fibrosis progression and that inhibition of miR-323a-3p may be a promising therapeutic target for the treatment of cardiac fibrosis.

MicroRNA-222 Promotes the Proliferation of Pulmonary Arterial Smooth Muscle Cells by Targeting P27 and TIMP3.

BACKGROUND/AIMS: Aberrant vascular smooth muscle cell (VSMC) proliferation plays an important role in the development of pulmonary artery hypertension (PAH). Dysregulated microRNAs (miRNAs, miRs) have been implicated in the progression of PAH. miR-222 has a pro-proliferation effect on VSMCs while it has an anti-proliferation effect on vascular endothelial cells (ECs). As the biological function of a single miRNA could be cell-type specific, the role of miR-222 in pulmonary artery smooth muscle cell (PASMC) proliferation is not clear and deserves to be explored. METHODS: PASMCs were transfected with miR-222 mimic or inhibitor and PASMC proliferation was determined by Western blot for PCNA, Ki-67 and EdU staining, and cell number counting. The target genes of miR-222 including P27 and TIMP3 were determined by luciferase assay and Western blot. In addition, the functional rescue experiments were performed based on miR-222 inhibitor and siRNAs to target genes. RESULTS: miR-222 mimic promoted PASMC proliferation while miR-222 inhibitor decreased that. TIMP3 was identified to be a direct target gene of miR-222 based on luciferase assay. Meanwhile, P27 and TIMP3 were up-regulated by miR-222 inhibitor and down-regulated by miR-222 mimic. Moreover, P27 siRNA and TIMP3 siRNA could both attenuate the anti-proliferation effect of miR-222 inhibitor in PASMCs, supporting that P27 and TIMP3 are at least partially responsible for the regulatory effect of miR-222 in PASMCs. CONCLUSION: miR-222 promotes PASMC proliferation at least partially through targeting P27 and TIMP3.

MicroRNA-181b Controls Atherosclerosis and Aneurysms Through Regulation of TIMP-3 and Elastin.

RATIONALE: Atherosclerosis and aneurysms are leading causes of mortality worldwide. MicroRNAs (miRs) are key determinants of gene and protein expression, and atypical miR expression has been associated with many cardiovascular diseases; although their contributory role to atherosclerotic plaque and abdominal aortic aneurysm stability are poorly understood. OBJECTIVE: To investigate whether miR-181b regulates tissue inhibitor of metalloproteinase-3 expression and affects atherosclerosis and aneurysms. METHODS AND RESULTS: Here, we demonstrate that miR-181b was overexpressed in symptomatic human atherosclerotic plaques and abdominal aortic aneurysms and correlated with decreased expression of predicted miR-181b targets, tissue inhibitor of metalloproteinase-3, and elastin. Using the well-characterized mouse atherosclerosis models of Apoe-/- and Ldlr-/-, we observed that in vivo administration of locked nucleic acid anti-miR-181b retarded both the development and the progression of atherosclerotic plaques. Systemic delivery of anti-miR-181b in angiotensin II-infused Apoe-/- and Ldlr-/- mice attenuated aneurysm formation and progression within the ascending, thoracic, and abdominal aorta. Moreover, miR-181b inhibition greatly increased elastin and collagen expression, promoting a fibrotic response and subsequent stabilization of existing plaques and aneurysms. We determined that miR-181b negatively regulates macrophage tissue inhibitor of metalloproteinase-3 expression and vascular smooth muscle cell elastin production, both important factors in maintaining atherosclerotic plaque and aneurysm stability. Validation studies in Timp3-/- mice confirmed that the beneficial effects afforded by miR-181b inhibition are largely tissue inhibitor of metalloproteinase-3 dependent, while also revealing an additional protective effect through elevating elastin synthesis. CONCLUSIONS: Our findings suggest that the management of miR-181b and its target genes provides therapeutic potential for limiting the progression of atherosclerosis and aneurysms and protecting them from rupture.

Identification of TIM3 2'-fluoro oligonucleotide aptamer by HT-SELEX for cancer immunotherapy.

TIM3 belongs to a family of receptors that are involved in T-cell exhaustion and Treg functions. The development of new therapeutic agents to block this type of receptors is opening a new avenue in cancer immunotherapy. There are currently several clinical trials ongoing to combine different immune-checkpoint blockades to improve the outcome of cancer patients. Among these combinations we should underline PD1:PDL1 axis and TIM3 blockade, which have shown very promising results in preclinical settings. Most of these types of therapeutic agents are protein cell-derived products, which, although broadly used in clinical settings, are still subject to important limitations. In this work we identify by HT-SELEX TIM3 non-antigenic oligonucleotide aptamers (TIM3Apt) that bind with high affinity and specificity to the extracellular motives of TIM3 on the cell surface. The TIM3Apt1 in its monomeric form displays a potent antagonist capacity on TIM3-expressing lymphocytes, determining the increase of IFN-γ secretion. In colon carcinoma tumor-bearing mice, the combinatorial treatment of TIM3Apt1 and PDL1-antibody blockade is synergistic with a remarkable antitumor effect. Immunotherapeutic aptamers could represent an attractive alternative to monoclonal antibodies, as they exhibit important advantages; namely, lower antigenicity, being chemically synthesized agents with a lower price of manufacture, providing higher malleability, and antidote availability.

A novel action mechanism for MPT0G013, a derivative of arylsulfonamide, inhibits tumor angiogenesis through up-regulation of TIMP3 expression.

Tissue inhibitors of metalloproteinases 3 (TIMP3) were originally characterized as inhibitors of matrix metalloproteinases (MMPs), acting as potent antiangiogenic proteins. In this study, we demonstrated that the arylsulfonamide derivative MPT0G013 has potent antiangiogenic activities in vitro and in vivo viainducing TIMP3 expression. Treatments with MPT0G013 significantly inhibited endothelial cell functions, such as cell proliferation, migration, and tube formation, as well as induced p21 and cell cycle arrest at the G0/G1 phase. Subsequent microarray analysis showed significant induction of TIMP3 gene expression by MPT0G013, and siRNA-mediated blockage of TIMP3 up-regulation abrogated the antiangiogenic activities of MPT0G013 and prevented inhibition of p-AKT and p-ERK proteins. Importantly, MPT0G013 exhibited antiangiogenic activities in in vivo Matrigel plug assays, inhibited tumor growth and up-regulated TIMP3 and p21 proteins in HCT116 mouse xenograft models. These data suggest potential therapeutic application of MPT0G013 for angiogenesis-related diseases such as cancer.

Sulfated glycosaminoglycans control the extracellular trafficking and the activity of the metalloprotease inhibitor TIMP-3.

Tissue inhibitor of metalloproteinase 3 (TIMP-3) is an important regulator of extracellular matrix (ECM) turnover. TIMP-3 binds to sulfated ECM glycosaminoglycans or is endocytosed by cells via low-density lipoprotein receptor-related protein 1 (LRP-1). Here, we report that heparan sulfate (HS) and chondroitin sulfate E (CSE) selectively regulate postsecretory trafficking of TIMP-3 by inhibiting its binding to LRP-1. HS and CSE also increased TIMP-3 affinity for glycan-binding metalloproteinases, such as adamalysin-like metalloproteinase with thrombospondin motifs 5 (ADAMTS-5), by reducing the dissociation rate constants. The sulfation pattern was crucial for these activities because monosulfated or truncated heparin had a reduced ability to bind to TIMP-3 and increase its affinity for ADAMTS-5. Therefore, sulfation of ECM glycans regulates the levels and inhibitory activity of TIMP-3 and modulates ECM turnover, and small mimicries of sulfated glycans may protect the tissue from the excess destruction seen in diseases such as osteoarthritis, cancer, and atherosclerosis.

Altered microRNA expression after infection with human cytomegalovirus leads to TIMP3 downregulation and increased shedding of metalloprotease substrates, including MICA.

Proteolytic shedding of ligands for the NK group 2D (NKG2D) receptor is a strategy used by tumors to modulate immune recognition by NK cells and cytotoxic T cells. A number of metalloproteases, especially those of the A disintegrin and metalloprotease (ADAM) family, can mediate NKG2D ligand cleavage and this process can be modulated by expression of the thiol isomerase ERp5. In this article, we describe that an increased shedding of the NKG2D ligand MICA is observed postinfection with several strains of human CMV due to an enhanced activity of ADAM17 (TNF-α converting enzyme) and matrix metalloprotease 14 caused by a reduction in the expression of the endogenous inhibitor of metalloproteases tissue inhibitors of metalloproteinase 3 (TIMP3). This decrease in TIMP3 expression correlates with increased expression of a cellular miRNA known to target TIMP3, and we also identify a human CMV-encoded microRNA able to modulate TIMP3 expression. These observations characterize a novel viral strategy to influence the shedding of cell-surface molecules involved in immune response modulation. They also provide an explanation for previous reports of increased levels of various ADAM17 substrates in the serum from patients with CMV disease. Consistent with this hypothesis, we detected soluble MICA in serum of transplant recipients with CMV disease. Finally, these data suggest that it might be worthwhile to prospectively study ADAM17 activity in a larger group of patients to assay whether this might be a useful biomarker to identify patients at risk for development of CMV disease.

Down-regulation of TIMP3 leads to increase in TACE expression and TNFα production by placental trophoblast cells.

PROBLEM: To determine whether down-regulation of TIMP3 expression promotes TACE expression and increases in TNFα production by placental trophoblast cells. METHOD OF STUDY: Placental expression of TIMP3 and TACE was examined by immunostaining and Western blot. Effects of TIMP3 on TACE expression and TNFα production were assessed by transfection of TIMP3 siRNA into trophoblasts isolated from normal placentas. Effects of oxidative stress on trophoblast TIMP3 expression and TNFα production were also determined. Trophoblast production of TIMP3, TACE and TNFα were measured by ELISA. RESULTS: TIMP3 expression was markedly reduced in preeclamptic placentas compared with normal placentas; oxidative stress down-regulated trophoblast TIMP3 expression and production, P < 0.01. Down-regulation of TIMP3 expression by TIMP3 siRNA resulted in significant increases in TACE expression and TNFα production, P < 0.01. CONCLUSION: As TIMP3 is an endogenous TACE inhibitor, down-regulation of trophoblast TIMP3 expression/activity could result in increased TACE expression and subsequently lead to increased TNFα production in preeclamptic placentas.

MicroRNA-191 correlates with poor prognosis of colorectal carcinoma and plays multiple roles by targeting tissue inhibitor of metalloprotease 3.

MicroRNA-191 (miR-191) is reported to be overexpressed in colorectal carcinoma (CRC), but the role of miR-191 in CRC progress remained unclear. This study demonstrated that High miR-191 expression was associated with clinical stage, lymph node metastasis, liver metastasis and depth of tumor invasion. Kaplan-Meier analysis indicated that patients with high miR-191 expression had a poor overall survival. Moreover, multivariate analysis showed that miR-191 was an independent prognostic factor in patients with CRC. Furthermore, we found that tissue inhibitor of metalloprotease 3 (TIMP3) was a direct target of miR-191 in colorectal cancer SW620 cells. TIMP3 downregulation mediated by miR-191 activated matrix metalloproteinases (MMPs) and thus promoted invasiveness of cancer cells. Anti-miR-191 could attenuate the invasiveness, suppress proliferation and induce apoptosis by restoring TIMP3 expression. Our results suggested that miR-191 might be a potential diagnostic and therapeutic target in patients with colorectal cancer.

The atypical mechanosensitive microRNA-712 derived from pre-ribosomal RNA induces endothelial inflammation and atherosclerosis.

MicroRNAs (miRNAs) regulate cardiovascular biology and disease, but the role of flow-sensitive microRNAs in atherosclerosis is still unclear. Here we identify miRNA-712 (miR-712) as a mechanosensitive miRNA upregulated by disturbed flow (d-flow) in endothelial cells, in vitro and in vivo. We also show that miR-712 is derived from an unexpected source, pre-ribosomal RNA, in an exoribonuclease-dependent but DiGeorge syndrome critical region 8 (DGCR8)-independent manner, suggesting that it is an atypical miRNA. Mechanistically, d-flow-induced miR-712 downregulates tissue inhibitor of metalloproteinase 3 (TIMP3) expression, which in turn activates the downstream matrix metalloproteinases (MMPs) and a disintegrin and metalloproteases (ADAMs) and stimulate pro-atherogenic responses, endothelial inflammation and permeability. Furthermore, silencing miR-712 by anti-miR-712 rescues TIMP3 expression and prevents atherosclerosis in murine models of atherosclerosis. Finally, we report that human miR-205 shares the same 'seed sequence' as murine-specific miR-712 and also targets TIMP3 in a flow-dependent manner. Targeting these mechanosensitive 'athero-miRs' may provide a new treatment paradigm in atherosclerosis.

Tissue inhibitor of metalloproteinases-3 peptides inhibit angiogenesis and choroidal neovascularization in mice.

Tissue inhibitors of metalloproteinases (TIMPs) while originally characterized as inhibitors of matrix metalloproteinases (MMPs) have recently been shown to have a wide range of functions that are independent of their MMP inhibitory properties. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is a potent inhibitor of VEGF-mediated angiogenesis and neovascularization through its ability to block the binding of VEGF to its receptor VEGFR-2. To identify and characterize the anti-angiogenic domain of TIMP-3, structure function analyses and synthetic peptide studies were performed using VEGF-mediated receptor binding, signaling, migration and proliferation. In addition, the ability of TIMP-3 peptides to inhibit CNV in a mouse model was evaluated. We demonstrate that the anti-angiogenic property resides in the COOH-terminal domain of TIMP-3 protein which can block the binding of VEGF specifically to its receptor VEGFR-2, but not to VEGFR-1 similar to the full-length wild-type protein. Synthetic peptides corresponding to putative loop 6 and tail region of TIMP-3 have anti-angiogenic properties as determined by inhibition of VEGF binding to VEGFR-2, VEGF-induced phosphorylation of VEGFR-2 and downstream signaling pathways as well as endothelial cell proliferation and migration in response to VEGF. In addition, we show that intravitreal administration of TIMP-3 peptide could inhibit the size of laser-induced choroidal neovascularization lesions in mice. Thus, we have identified TIMP-3 peptides to be efficient inhibitors of angiogenesis and have a potential to be used therapeutically in diseases with increased neovascularization.

Regulation of Th1/Th2 polarization by tissue inhibitor of metalloproteinase-3 via modulating dendritic cells.

Tissue inhibitor of metalloproteinase-3 (TIMP-3) is one of a family of proteins inhibiting matrix metalloproteinases, which has also been identified as a mediator for checking inflammation. Meanwhile, it is well known that inflammation causes the activation of the immune response. However, it is not clear whether TIMP-3 plays a role in the immune system. In the present study, we demonstrated a novel function of TIMP-3 in Th1/Th2 polarization through its influence on the antigen-presenting cells. First, TIMP-3 was found strikingly up-regulated by IL-4 during the differentiation of human dendritic cells via the p38MAPK pathway. Second, the expression of costimulatory molecule-CD86 was repressed by TIMP-3. Besides, the induction of IL-12 in matured dendritic cells was significantly inhibited in a PI3K-dependent manner. Furthermore, dendritic cells matured in the presence of TIMP-3 could stimulate allogeneic naive T helper (Th) cells to display a prominent Th2 polarization. Importantly, in an autoimmune disorder-primary immune thrombocytopenia, TIMP-3 showed a statistically positive correlation with IL-4 and platelet count, but a negative correlation with IFN-γ in patient blood samples. Collectively, these in vitro and in vivo data clearly suggested a novel role of TIMP-3 in Th1/Th2 balance in humans.

P14ARF inhibits human glioblastoma-induced angiogenesis by upregulating the expression of TIMP3.

Malignant gliomas are the most common and the most lethal primary brain tumors in adults. Among malignant gliomas, 60%-80% show loss of P14ARF tumor suppressor activity due to somatic alterations of the INK4A/ARF genetic locus. The tumor suppressor activity of P14ARF is in part a result of its ability to prevent the degradation of P53 by binding to and sequestering HDM2. However, the subsequent finding of P14ARF loss in conjunction with TP53 gene loss in some tumors suggests the protein may have other P53-independent tumor suppressor functions. Here, we report what we believe to be a novel tumor suppressor function for P14ARF as an inhibitor of tumor-induced angiogenesis. We found that P14ARF mediates antiangiogenic effects by upregulating expression of tissue inhibitor of metalloproteinase-3 (TIMP3) in a P53-independent fashion. Mechanistically, this regulation occurred at the gene transcription level and was controlled by HDM2-SP1 interplay, where P14ARF relieved a dominant negative interaction of HDM2 with SP1. P14ARF-induced expression of TIMP3 inhibited endothelial cell migration and vessel formation in response to angiogenic stimuli produced by cancer cells. The discovery of this angiogenesis regulatory pathway may provide new insights into P53-independent P14ARF tumor-suppressive mechanisms that have implications for the development of novel therapies directed at tumors and other diseases characterized by vascular pathology.

HMGB1 attenuates cardiac remodelling in the failing heart via enhanced cardiac regeneration and miR-206-mediated inhibition of TIMP-3.

AIMS: HMGB1 injection into the mouse heart, acutely after myocardial infarction (MI), improves left ventricular (LV) function and prevents remodeling. Here, we examined the effect of HMGB1 in chronically failing hearts. METHODS AND RESULTS: Adult C57 BL16 female mice underwent coronary artery ligation; three weeks later 200 ng HMGB1 or denatured HMGB1 (control) were injected in the peri-infarcted region of mouse failing hearts. Four weeks after treatment, both echocardiography and hemodynamics demonstrated a significant improvement in LV function in HMGB1-treated mice. Further, HMGB1-treated mice exhibited a ∼23% reduction in LV volume, a ∼48% increase in infarcted wall thickness and a ∼14% reduction in collagen deposition. HMGB1 induced cardiac regeneration and, within the infarcted region, it was found a ∼2-fold increase in c-kit⁺ cell number, a ∼13-fold increase in newly formed myocytes and a ∼2-fold increase in arteriole length density. HMGB1 also enhanced MMP2 and MMP9 activity and decreased TIMP-3 levels. Importantly, miR-206 expression 3 days after HMGB1 treatment was 4-5-fold higher than in control hearts and 20-25 fold higher that in sham operated hearts. HMGB1 ability to increase miR-206 was confirmed in vitro, in cardiac fibroblasts. TIMP3 was identified as a potential miR-206 target by TargetScan prediction analysis; further, in cultured cardiac fibroblasts, miR-206 gain- and loss-of-function studies and luciferase reporter assays showed that TIMP3 is a direct target of miR-206. CONCLUSIONS: HMGB1 injected into chronically failing hearts enhanced LV function and attenuated LV remodelling; these effects were associated with cardiac regeneration, increased collagenolytic activity, miR-206 overexpression and miR-206 -mediated inhibition of TIMP-3.