Enhanced angiogenesis and reduced contraction in thrombospondin-2-null wounds is associated with increased levels of matrix metalloproteinases-2 and -9, and soluble VEGF.

Thrombospondin-2 (TSP2) is an inhibitor of angiogenesis with pro-apoptotic and anti-proliferative effects on endothelial cells. Mice deficient in this matricellular protein display improved recovery from ischemia and accelerated wound healing associated with alterations in angiogenesis and extracellular matrix remodeling. In this study, we probed the function of TSP2 by performing a detailed analysis of dermal wounds and wound-derived fibroblasts. Specifically, we analyzed incisional wounds by tensiometry and found no differences in strength recovery between wild-type and TSP2-null mice. In addition, analysis of full-thickness excisional wounds by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end labeling stain and MIB-5 immunohistochemistry revealed similar numbers of apoptotic and proliferating cells, respectively. In contrast, the levels of matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitors of metalloproteinase (TIMP)-1, TIMP-2, and soluble vascular endothelial growth factor were increased in wounds of TSP2-null mice. Evaluation of the ability of TSP2-null wound fibroblasts to contract collagen gels revealed that it was compromised, even though TSP2-null wounds displayed normal myofibroblast content. Therefore, we conclude that the lack of TSP2 leads to aberrant extracellular matrix remodeling, increased neovascularization, and reduced contraction due in part to elevated levels of MMP-2 and MMP-9. These observations provide in vivo supporting evidence for a newly proposed function of TSP2 as a modulator of extracellular matrix remodeling.

Elevated expression of isopeptide bond cross-links contributes to fibrosis in scleroderma and the healing wounds of tight skin mice.

Scleroderma is a chronic disease characterized by excessive tissue fibrosis. Recent studies indicate that cultured dermal fibroblasts isolated from patients produce excessive amounts of collagen and other extracellular matrix components. In this study, we investigated the mechanism(s) of abnormal extracellular matrix accumulation in the scleroderma biopsies and the healing wounds of Tsk1/+ mice. Full-thickness excisional wounds were made in Tsk1/+ and wild-type mice and were subsequently harvested at days 7, 10, and 14 postinjury. The levels of pro-fibrotic cytokine, transforming growth factor were elevated in the wounds of Tsk1/+ mice. Interestingly, the levels of matrix metalloproteinase were significantly reduced in the granulation tissue of Tsk1/+ mice in comparison with wild-type. Furthermore, immunohistochemical analysis of the wounds indicated that the levels of gamma-glutamyl-epsilon-lysine cross-links were elevated in the granulation tissue of Tsk1/+ mice as well as the fibrotic lesions of scleroderma specimens. Collectively, these findings indicate that elevated collagen synthesis and decreased matrix metalloproteinase levels, in combination with increased isopeptide bond cross-links, contribute to abnormal collagen synthesis and assembly in granulation tissue of Tsk1/+ mice and the fibrotic lesions of scleroderma patients.

The role of thrombospondins 1 and 2 in the regulation of cell-matrix interactions, collagen fibril formation, and the response to injury.

Thrombospondins (TSPs) 1 and 2 are extracellular modular glycoproteins that are best known for their anti-angiogenic properties and their ability to modulate cell-matrix interactions. However, these proteins, and in particular TSP2, are pleiotropic in function and affect processes as disparate as bone growth and hemostasis. In recognition of their ability to influence a wide variety of cell functions, and in the absence of convincing evidence for their participation as integral components of extracellular structures, the term 'matricellular' has been applied to these and a small group of functionally related proteins. In this review, we focus on the role of TSP1 and 2 in two forms of injury in mice, excisional skin wounds and subcutaneously implanted biomaterials, and take advantage of mice with targeted disruptions of one or both genes to identify likely biochemical mechanisms that could account for the characteristics of the injury response in these knockout mice. In work that stems largely from our own laboratory, we show that pericellular levels of the matrix metalloproteinase, MMP2, are controlled to a large extent by TSP2 (and potentially also by TSP1), and that elevated levels of MMP2 are likely to account in part for defects as diverse as reduced cellular adhesion, abnormal collagen fibril structure, and increased endothelial cell and vascular proliferation.

Thrombospondin 2 levels are increased in aged mice: consequences for cutaneous wound healing and angiogenesis.

The inhibitor of angiogenesis, thrombospondin 2 (TSP2), belongs to a group of matricellular proteins that are induced in response to injury and modulate the healing of dermal wounds. Thus, TSP-2-null mice display abnormal connective tissue architecture and increased angiogenesis in the dermis, and heal wounds at an accelerated rate. In this study, we report that the content of TSP2 is increased in the uninjured skin of aged mice. Furthermore, in primary dermal fibroblasts, TSP2 expression is increased both as a function of the age of the donor and days in culture. To determine the significance of the increased TSP2 in aged mice (two years or older), we performed full-thickness excisional wounds and compared their healing in aged and young (3-4 months) wild-type and TSP2-null mice. Gross morphological examination of wounds indicated that aged TSP2-null mice healed faster than their aged wild-type counterparts, but healing in aged mice was always sub-optimal in comparison to that in young animals. Surprisingly, despite the increase in TSP2, a potent inhibitor of angiogenesis, in wounds in aged mice, the vascular density of these wounds was not reduced in comparison to that in young animals. However, immunohistochemical analysis of healing wounds revealed a shift in the peak content of TSP2, from day 10 in young mice to day 14 or later in aged mice, and there was a corresponding delay in the expected increase in matrix metalloproteinase (MMP) 2 levels in aged TSP2-null mice. We suggest that the delay in expression of TSP2 and MMP2 in the wounds of aged mice could contribute to their impaired rate of wound healing.

Proteolysis of cell-surface tissue transglutaminase by matrix metalloproteinase-2 contributes to the adhesive defect and matrix abnormalities in thrombospondin-2-null fibroblasts and mice.

Thrombospondin (TSP)-2-null dermal fibroblasts display an attachment defect that results from increased matrix metalloproteinase (MMP)-2 levels in their conditioned media. To investigate the molecular mechanisms responsible for this defect, we analyzed the activity of tissue transglutaminase (tTG) in TSP-2-null dermal fibroblasts and in tissues of TSP-2-null mice. tTG functions as a co-receptor for beta1 and beta3 integrins and stabilizes extracellular matrix proteins by introduction of isopeptide cross-links. Cell-surface tTG activity was reduced in TSP-2-null cells (0.50 +/- 0.05 arbitrary units versus 0.84 +/- 0.07 for wild type; P < or = 0.05), and addition of MMP-2 to the culture medium of wild-type cells caused a 35% reduction in cell-surface tTG activity. tTG was susceptible to proteolysis by MMP-2 in vitro, and addition of the MMP inhibitor TIMP-2 to TSP-2-null cells restored tTG activity (0.3 +/- 0.08 for untreated cells; 0.71 +/- 0.09 with TIMP-2). TSP-2-null mice had reduced tTG activity in skin, as measured by incorporation of fluorescein isothiocyanate-labeled cadaverine, and a threefold increase in acetic acid-extracted dermal collagen. Furthermore, isopeptide cross-links were reduced in both uninjured skin and in excisional wounds of TSP-2-null mice, as determined by morphometric immunohistochemical analysis, indicating that isopeptide cross-links are important for the stabilization of the collagenous matrix in dermis. These findings provide a mechanism for the reduced adhesion of TSP-2-null fibroblasts and an explanation for the increased collagen solubility and fragility of TSP-2-null skin.

Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis.

The establishment of neural circuitry requires vast numbers of synapses to be generated during a specific window of brain development, but it is not known why the developing mammalian brain has a much greater capacity to generate new synapses than the adult brain. Here we report that immature but not mature astrocytes express thrombospondins (TSPs)-1 and -2 and that these TSPs promote CNS synaptogenesis in vitro and in vivo. TSPs induce ultrastructurally normal synapses that are presynaptically active but postsynaptically silent and work in concert with other, as yet unidentified, astrocyte-derived signals to produce functional synapses. These studies identify TSPs as CNS synaptogenic proteins, provide evidence that astrocytes are important contributors to synaptogenesis within the developing CNS, and suggest that TSP-1 and -2 act as a permissive switch that times CNS synaptogenesis by enabling neuronal molecules to assemble into synapses within a specific window of CNS development.

The lack of thrombospondin-1 (TSP1) dictates the course of wound healing in double-TSP1/TSP2-null mice.

Thrombospondin (TSP) 1 and 2, share the same overall structure and interact with a number of the same cell-surface receptors. In an attempt to elucidate their biological roles more clearly, we generated double-TSP1/TSP2-null animals and compared their phenotype to those of TSP1- and TSP2-null mice. Double-null mice exhibited an apparent phenotype that primarily represented the sum of the abnormalities observed in the single-null mice. However, surprisingly, the wound-healing response in double-null mice resembled that in TSP1-null animals and differed from that in TSP2-nulls. Thus, although the excisional wounds of TSP2-null mice are characterized by increased neovascularization and heal at an accelerated rate, TSP1-null and double-null animals demonstrated delayed healing, as indicated by the prolonged persistence of inflammation and delayed scab loss. Immunohistochemical analysis showed that, similar to TSP1-null mice, the granulation tissue of double-null mice was not excessively vascularized. Furthermore as in TSP1-nulls, decreases in macrophage recruitment and in the levels of monocyte chemoattractant protein-1 indicated that the inflammatory phase of the wound-healing response was impaired in double-null mice. Our data demonstrate that the consequences of a lack of TSP1 predominate in the response of double-null mice, and dictate the course of wound healing. These findings reflect distinct temporal and spatial expressions of TSP1 and TSP2 in the healing wound.

Thrombospondin 2 deficiency in pregnant mice results in premature softening of the uterine cervix.

The gradual disorganization of collagen fibers in the stromal connective tissue of the uterine cervix is characteristic of progressive cervical softening during pregnancy. A lack of thrombospondin (TSP) 2 has been shown to be associated with altered collagen fibril morphology of connective-tissue-rich organs such as skin and tendon. The goal of this study was to determine the role of TSP2 in cervical softening by studying a TSP2-null mouse line. Creep testing showed that, in the nonpregnant animal and on Day 10 of pregnancy, there was no difference between the cervical extensibility of the wild-type and the TSP2-deficient mice. However, by Day 14 of pregnancy, the TSP2-null mice showed 4.5-fold increase in cervical extensibility, and by Day 18, a 6.1-fold increase, when compared with wild-type mice. A further indicator of compromised cervical integrity was that, on Days 14 and 18 of pregnancy, the cervix of TSP2-null mice broke rapidly under standard loading conditions that did not break the cervix of wild-type mice. Western blotting showed that TSP2 was expressed in the cervix of mice on Days 14 and 18 of pregnancy but not on Day 10 or in the nonpregnant animal. As determined by immunohistochemistry, the amount of matrix metalloproteinase 2 (MMP2) in the cervix of TSP2-null mice increased 11-fold on Day 14 of pregnancy and 19-fold on Day 18. Thus, TSP2-null mice provide an animal model to assist in the understanding of the molecular basis of spontaneous, premature softening of the uterine cervix.