Mice with tissue inhibitor of metalloproteinases 4 (Timp4) deletion succumb to induced myocardial infarction but not to cardiac pressure overload.

Tissue inhibitor of metalloproteinases 4 (TIMP4) is expressed highly in heart and found dysregulated in human cardiovascular diseases. It controls extracellular matrix remodeling by inhibiting matrix metalloproteinases (MMPs) and is implicated in processes including cell proliferation, apoptosis, and angiogenesis. Timp4-deficient mice (Timp4(-/-)) were generated to assess TIMP4 function in normal development and in models of heart disease. We deleted exons 1-3 of the Timp4 gene by homologous recombination. Timp4(-/-) mice are born healthy, develop normally, and produce litters of normal size and gender distribution. These mice show no compensation by overexpression of Timp1, Timp2, or Timp3 in the heart. Following cardiac pressure overload by aortic banding, Timp4(-/-) mice have comparable survival rate, cardiac histology, and cardiac function to controls. In this case, Timp4 deficiency is compensated by increased cardiac Timp2 expression. Strikingly, the induction of myocardial infarction (MI) leads to significantly increased mortality in Timp4(-/-) mice primarily due to left ventricular rupture. The post-MI mortality of Timp4(-/-) mice is reduced by administration of a synthetic MMP inhibitor. Furthermore, combining the genetic deletion of Mmp2 also rescues the higher post-MI mortality of Timp4(-/-) mice. Finally, Timp4(-/-) mice suffer reduced cardiac function at 20 months of age. Timp4 is not essential for murine development, although its loss moderately compromises cardiac function with aging. Timp4(-/-) mice are more susceptible to MI but not to pressure overload, and TIMP4 functions in its capacity as a metalloproteinase inhibitor after myocardial infarction.

First-in-Humans Study of 68Ga-DOTA-Siglec-9, a PET Ligand Targeting Vascular Adhesion Protein 1.

Sialic acid-binding immunoglubulinlike lectin 9 (Siglec-9) is a ligand of vascular adhesion protein 1. A 68Ga-labeled peptide of Siglec-9, 68Ga-DOTA-Siglec-9, holds promise as a novel PET tracer for imaging of inflammation. This first-in-humans study investigated the safety, tolerability, biodistribution, and radiation dosimetry of this radiopharmaceutical. Methods: Six healthy men underwent dynamic whole-body PET/CT. Serial venous blood samples were drawn from 1 to 240 min after intravenous injection of 162 ± 4 MBq of 68Ga-DOTA-Siglec-9. In addition to γ-counting, the plasma samples were analyzed by high-performance liquid chromatography to detect intact tracer and radioactive metabolites. Radiation doses were calculated using the OLINDA/EXM software, version 2.2. In addition, a patient with early rheumatoid arthritis was studied with both 68Ga-DOTA-Siglec-9 and 18F-FDG PET/CT to determine the ability of the new tracer to detect arthritis. Results:68Ga-DOTA-Siglec-9 was well tolerated by all subjects. 68Ga-DOTA-Siglec-9 was rapidly cleared from the blood circulation, and several radioactive metabolites were detected. The organs with the highest absorbed doses were the urinary bladder wall (0.38 mSv/MBq) and kidneys (0.054 mSv/MBq). The mean effective dose was 0.022 mSv/MBq (range, 0.020-0.024 mSv/MBq). Most importantly, however, 68Ga-DOTA-Siglec-9 was comparable to 18F-FDG in detecting arthritis. Conclusion: Intravenous injection of 68Ga-DOTA-Siglec-9 was safe and biodistribution was favorable for testing of the tracer in larger group of patients with rheumatoid arthritis, as is planned for the next phase of clinical trials. The effective radiation dose of 68Ga-DOTA-Siglec-9 was within the same range as the effective radiation doses of other 68Ga-labeled tracers. Injection of 150 MBq of 68Ga-DOTA-Siglec-9 would expose a subject to 3.3 mSv. These findings support the possible repeated clinical use of 68Ga-DOTA-Siglec-9, such as in trials to elucidate the treatment efficacy of novel drug candidates.

Mice with a deletion in the first intron of the Col1a1 gene develop age-dependent aortic dissection and rupture.

The functional significance of the first intron of the Col1a1 gene in regulation of type I collagen synthesis remains uncertain. A previous study in mice established that a mutated Col1a1 allele that lacked a large fraction of the first intron, but retained the sequences required for normal splicing, was subject to an age- and tissue-dependent decrease in expression. In this study, we report that mice homozygous for this deletion are predisposed to dissection and rupture of the aorta during their adult life. Aortic dissection was not detected in autopsies of heterozygous animals or their littermate controls. Electron micrographs revealed fewer collagen fibrils and less compacted, irregular elastic lamellae in the aortic walls of homozygous mutant animals. Northern analysis of aortic RNA from 2.5- and 12-month-old homozygous mutant mice revealed that Col1a1 mRNA levels were decreased by 29% and 42%, respectively, relative to those of control littermates. In 12-month-old heterozygotes, the decrease was 32%. Allele-specific amplification of heterozygous cDNAs demonstrated that this reduction was limited to transcripts from the mutant allele. The collagen content of the aortas of homozygous mutant mice was also significantly lower in comparison to that of age-matched, control animals. These data establish that the integrity of the aortic wall depends on an adequate content of type I collagen, and that continued synthesis of collagen in the aorta as a function of age is critically dependent on sequences in the first intron of the Col1a1 gene.

Characterization of the murine Timp4 gene, localization within intron 5 of the synapsin 2 gene and tissue distribution of the mRNA.

Tissue inhibitor of matrix metalloproteinases type 4 (TIMP-4), the newest member in the mammalian TIMP family of inhibitors of matrix metalloproteinases (MMPs), differs from the other three TIMPs by its restricted expression pattern. This suggests that TIMP-4 could play a role in tissue-specific regulation of extracellular matrix (ECM) turnover. To define this role, modulation of TIMP-4 production by overexpression, aberrant expression and inactivation of the Timp4 gene in transgenic mice should be performed. In preparation for such experiments we have cloned and characterized the murine Timp4 gene and determined the tissue distribution of its mRNA in mice. The gene spans 7.1 kb, consists of five exons and shares considerable homology with the other Timp genes. The gene is located on mouse chromosome 6 in an antisense orientation between exons 5 and 6 of the mouse synapsin 2 (Syn2) gene. A similar organization is common to all four human/mouse TIMP and SYN genes and to the single synapsin/Timp locus in Drosophila. The highest levels of TIMP-4 mRNA were seen in postnatal mouse heart, ovary and brain. Determination of the spatial expression pattern of TIMP-4 mRNA by in situ hybridization in the heart revealed a diffuse distribution in cardiac muscle cells. In the ovary, cyclic variation was observed in TIMP-4 mRNA levels. In situ hybridization demonstrated the strongest expression of TIMP-4 mRNA in the corpus luteum. The data suggest that TIMP-4 plays a role in the normal physiology of the heart and the ovary, most likely related to maintenance of the delicate balance between MMPs and TIMPs.

Osteoclast-specific cathepsin K deletion stimulates S1P-dependent bone formation.

Cathepsin K (CTSK) is secreted by osteoclasts to degrade collagen and other matrix proteins during bone resorption. Global deletion of Ctsk in mice decreases bone resorption, leading to osteopetrosis, but also increases the bone formation rate (BFR). To understand how Ctsk deletion increases the BFR, we generated osteoclast- and osteoblast-targeted Ctsk knockout mice using floxed Ctsk alleles. Targeted ablation of Ctsk in hematopoietic cells, or specifically in osteoclasts and cells of the monocyte-osteoclast lineage, resulted in increased bone volume and BFR as well as osteoclast and osteoblast numbers. In contrast, targeted deletion of Ctsk in osteoblasts had no effect on bone resorption or BFR, demonstrating that the increased BFR is osteoclast dependent. Deletion of Ctsk in osteoclasts increased their sphingosine kinase 1 (Sphk1) expression. Conditioned media from Ctsk-deficient osteoclasts, which contained elevated levels of sphingosine-1-phosphate (S1P), increased alkaline phosphatase and mineralized nodules in osteoblast cultures. An S1P1,3 receptor antagonist inhibited these responses. Osteoblasts derived from mice with Ctsk-deficient osteoclasts had an increased RANKL/OPG ratio, providing a positive feedback loop that increased the number of osteoclasts. Our data provide genetic evidence that deletion of CTSK in osteoclasts enhances bone formation in vivo by increasing the generation of osteoclast-derived S1P.

Tissue inhibitor of metalloproteinases 4 (TIMP4) is involved in inflammatory processes of human cardiovascular pathology.

Tissue inhibitors of matrix metalloproteinases (TIMPs) comprise a family of four members, of which TIMP4 is characterized by being primarily restricted to cardiovascular structures. We demonstrate with immunohistochemical analysis of healthy human tissue that TIMP4 is present in medial smooth muscle cells and adventitial capillaries of arteries as well as in cardiomyocytes. Animal studies have suggested a role for TIMP4 in several inflammatory diseases and cardiovascular pathologies. We therefore examined whether TIMP4 is involved in human inflammatory cardiovascular disorders, specifically atherosclerosis, giant cell arteritis and chronic rejection of heart allografts. TIMP4 was most clearly visible in cardiovascular tissue areas populated by abundant inflammatory cells, mainly macrophages and CD3+ T cells. Using western blotting and immunocytochemistry, human blood derived lymphocytes, monocytes/macrophages and mast cells were shown to produce TIMP4. In advanced atherosclerotic lesions, TIMP4 was detected around necrotic lipid cores, whereas TIMP3 and caspase 3 resided within and around the core regions, indicating different roles for TIMP3 and TIMP4 in inflammation-induced apoptosis and in matrix turnover. In conclusion, the data demonstrate upregulation of TIMP4 in human cardiovascular disorders exhibiting inflammation, suggesting its future use as a novel systemic marker for vascular inflammation.

Individual Timp deficiencies differentially impact pro-MMP-2 activation.

Membrane-type matrix metalloproteinases (MT-MMPs) have emerged as key enzymes in tumor cell biology. The importance of MT1-MMP, in particular, is highlighted by its ability to activate pro-MMP-2 at the cell surface through the formation of a trimolecular complex comprised of MT1-MMP/tissue inhibitor of metalloproteinase-2 (TIMP-2)/pro-MMP-2. TIMPs 1-4 are physiological MMP inhibitors with distinct roles in the regulation of pro-MMP-2 processing. Here, we have shown that individual Timp deficiencies differentially affect MMP-2 processing using primary mouse embryonic fibroblasts (MEFs). Timp-3 deficiency accelerated pro-MMP-2 activation in response to both cytochalasin D and concanavalin A. Exogenous TIMP-2 and N-TIMP-3 inhibited this activation, whereas TIMP-3 containing matrix from wild-type MEFs did not rescue the enhanced MMP-2 activation in Timp-3(-/-) cells. Increased processing of MMP-2 did not arise from increased expression of MT1-MMP, MT2-MMP, or MT3-MMP or altered expression of TIMP-2 and MMP-2. To test whether increased MMP-2 processing in Timp-3(-/-) MEFs is dependent on TIMP-2, double deficient Timp-2(-/-)/-3(-/-) MEFs were used. In these double deficient cells, the cleavage of pro-MMP-2 to its intermediate form was substantially increased, but the subsequent cleavage of intermediate-MMP-2 to fully active form, although absent in Timp-2(-/-) MEFs, was detectable with combined Timp-2(-/-)/-3(-/-) deficiency. TIMP-4 associates with MMP-2 and MT1-MMP in a manner similar to TIMP-3, but its deletion had no effect on pro-MMP-2 processing. Thus, TIMP-3 provides an inherent regulation over the kinetics of pro-MMP-2 processing, serving at a level distinct from that of TIMP-2 and TIMP-4.