Thrombospondin-2 regulates extracellular matrix production, LOX levels, and cross-linking via downregulation of miR-29.

Collagen fibrillogenesis and crosslinking have long been implicated in extracellular matrix (ECM)-dependent processes such as fibrosis and scarring. However, the extent to which matricellular proteins influence ECM protein production and fibrillar collagen crosslinking has yet to be determined. Here we show that thrombospondin 2 (TSP2), an anti-angiogenic matricellular protein, is an important modulator of ECM homeostasis. Specifically, through a fractionated quantitative proteomics approach, we show that loss of TSP2 leads to a unique ECM phenotype characterized by a significant decrease in fibrillar collagen, matricellular, and structural ECM protein production in the skin of TSP2 KO mice. Additionally, TSP2 KO skin displays decreased lysyl oxidase (LOX), which manifests as an increase in fibrillar collagen solubility and decreased levels of LOX-mediated fibrillar collagen crosslinking. We show that these changes are indirectly mediated by miR-29, a major regulator of ECM proteins and LOX, as miR-29 expression is increased in the TSP2 KO. Altogether, these findings indicate that TSP2 contributes to ECM production and assembly by regulating miR-29 and LOX.

A hidden structural vulnerability in the thrombospondin-2 deficient aorta increases the propensity to intramural delamination.

Mice lacking thrombospondin-2 (TSP2) represent an animal model of impaired collagen fibrillogenesis. Collagen constitutes ~1/3 of the wall of the normal murine descending thoracic aorta (DTA) and is thought to confer mechanical strength at high pressures. Microstructural analysis of the DTA from TSP2-null mice revealed irregular and disorganized collagen fibrils in the adventitia and at the interface between the media and adventitia. Yet, biaxial mechanical tests performed under physiologic loading conditions showed that most mechanical metrics, including stress and stiffness, were not different between mutant and control DTAs at 20- and 40-weeks of age, thus suggesting that the absence of TSP2 is well compensated under normal conditions. A detailed bilayered analysis of the wall mechanics predicted, however, that the adventitia of TSP2-null DTAs fails to engage at high pressures, which could render the media vulnerable to mechanical damage. Failure tests confirmed that the pressure at which the DTA ruptures is significantly lower in 20-week-old TSP2-null mice compared to age-matched controls (640±37 vs. 1120±45mmHg). Moreover, half of the 20-week-old and all 40-week-old mutant DTAs failed by delamination, not rupture. This delamination occurred at the interface between the media and the adventitia, with separation planes often observed at ~45 degrees with respect to the circumferential/axial directions. Combined with the observed microstructural anomalies, our theoretical-experimental biomechanical results suggest that TSP2-null DTAs are more susceptible to material failure when exposed to high pressures and this vulnerability may result from a reduced resistance to shear loading at the medial/adventitial border.

The host response to naturally-derived extracellular matrix biomaterials.

Biomaterials based on natural materials including decellularized tissues and tissue-derived hydrogels are becoming more widely used for clinical applications. Because of their native composition and structure, these biomaterials induce a distinct form of the foreign body response that differs from that of non-native biomaterials. Differences include direct interactions with cells via preserved moieties as well as the ability to undergo remodeling. Moreover, these biomaterials could elicit adaptive immune responses due to the presence of modified native molecules. Therefore, these biomaterials present unique challenges in terms of understanding the progression of the foreign body response. This review covers this response to natural materials including natural polymers, decellularized tissues, cell-derived matrix, tissue derived hydrogels, and biohybrid materials. With the expansion of the fields of regenerative medicine and tissue engineering, the current repertoire of biomaterials has also expanded and requires continuous investigation of the responses they elicit.

Cryopreserved human amniotic membrane and a bioinspired underwater adhesive to seal and promote healing of iatrogenic fetal membrane defect sites.

INTRODUCTION: We investigated the ability of cryopreserved human amniotic membrane (hAM) scaffold sealed with an underwater adhesive, bio-inspired by marine sandcastle worms to promote healing of iatrogenic fetal membrane defects in a pregnant swine model. METHODS: Twelve Yucatan miniature pigs underwent laparotomy under general anesthesia at 70 days gestation (term = 114 days). The gestational sacs were assigned to uninstrumented (n = 24) and instrumented with 12 Fr trocar, which was further randomized into four different arms-no hAM patch, (n = 22), hAM patch secured with suture (n = 16), hAM patch with no suture (n = 14), and hAM patch secured with adhesive (n = 9). The animals were euthanized 20 days after the procedure. Gross and histological examination of the entry site was performed for fetal membrane healing. RESULTS: There were no differences in fetal survival, amniotic fluid levels, or dye-leakage from the amniotic cavity between the groups. The fetal membranes spontaneously healed in instrumented sacs without hAM patches. In sacs with hAM patches secured with sutures, the patch was incorporated into the swine fetal membranes. In sacs with hAM patches without sutures, 100% of the patches were displaced from the defect site, whereas in sacs with hAM patches secured with adhesive 55% of the patches remained in place and showed complete healing (p = 0.04). DISCUSSION: In contrast to humans, swine fetal membranes heal spontaneously after an iatrogenic injury and thus not an adequate model. hAM patches became incorporated into the defect site by cellular ingrowth from the fetal membranes. The bioinspired adhesive adhered the hAM patches within the defect site.

Altered extracellular matrix remodeling and angiogenesis in sponge granulomas of thrombospondin 2-null mice.

The matricellular angiogenesis inhibitor, thrombospondin (TSP) 2, has been shown to be an important modulator of wound healing and the foreign body response. Specifically, TSP2-null mice display improved healing with minimal scarring and form well-vascularized foreign body capsules. In this study we performed subcutaneous implantation of sponges and investigated the resulting angiogenic and fibrogenic responses. Histological and immunohistochemical analysis of sponges, excised at 7, 14, and 21 days after implantation, revealed significant differences between TSP2-null and wild-type mice. Most notably, TSP2-null mice exhibited increased angiogenesis and fibrotic encapsulation of the sponge. However, invasion of dense tissue was compromised, even though its overall density was increased. Furthermore, histomorphometry and biochemical assays demonstrated a significant increase in the extracellular distribution of matrix metalloproteinase (MMP) 2, but no change in the levels of active transforming growth factor-beta(1). The alterations in neovascularization, dense tissue invasion, and MMP2 in TSP2-null mice coincided with the deposition of TSP2 in the extracellular matrix of wild-type animals. These observations support the proposed role of TSP2 as a modulator of angiogenesis and matrix remodeling during tissue repair. In addition, they provide in vivo evidence for a newly proposed function of TSP2 as a modulator of extracellular MMP2 levels.

A mouse knock-in model exposes sequential proteolytic pathways that regulate p27Kip1 in G1 and S phase.

The protein p27Kip1 is an inhibitor of cell division. An increase in p27 causes proliferating cells to exit from the cell cycle, and a decrease in p27 is necessary for quiescent cells to resume division. Abnormally low amounts of p27 are associated with pathological states of excessive cell proliferation, especially cancers. In normal and tumour cells, p27 is regulated primarily at the level of translation and protein turnover. Phosphorylation of p27 on threonine 187 (T187) by cyclin-dependent kinase 2 (Cdk2) is thought to initiate the major pathway for p27 proteolysis. To critically test the importance of this pathway in vivo, we replaced the murine p27 gene with one that encoded alanine instead of threonine at position 187 (p27T187A). Here we show that cells expressing p27T187A were unable to downregulate p27 during the S and G2 phases of the cell cycle, but that this had a surprisingly modest effect on cell proliferation both in vitro and in vivo. Our efforts to explain this unexpected result led to the discovery of a second proteolytic pathway for controlling p27, one that is activated by mitogens and degrades p27 exclusively during G1.

Regulation of angiogenesis and matrix remodeling by localized, matrix-mediated antisense gene delivery.

Implantation of biomaterials, such as glucose sensors, leads to the formation of a poorly vascularized collagenous capsule that can lead to implant failure. This process, known as the foreign body reaction (FBR), develops in response to almost all biomaterials and consists of overlapping phases similar to those in wound healing. Implantation of porous biomaterials, such as polyvinyl alcohol sponges, also leads to granuloma formation within the interstices of the sponge prior to encapsulation by the FBR. We asked whether delivery of an antisense cDNA for the potent angiogenesis inhibitor thrombospondin (TSP) 2 would enhance blood vessel formation and alter collagen fibrillogenesis in the sponge granuloma and capsule. Collagen solutions were mixed with plasmid to generate gene-activated matrices (GAMs) and applied to biomaterials that were then implanted subcutaneously. Sustained expression of plasmid-encoded proteins was observed at 2 weeks and a month following implantation. In vivo delivery of plasmids, encoding either sense or antisense TSP2 cDNA, altered blood vessel formation and collagen deposition in TSP2-null and wild-type mice, respectively. Untreated implants, implanted next to GAM-treated implants, did not show exogenous gene expression and did not elicit altered responses, suggesting that gene delivery was limited to implant sites. This method of antisense DNA delivery has the potential to improve the performance and life span of implantable delivery devices and biosensors.

Thrombospondin-2 plays a protective role in multistep carcinogenesis: a novel host anti-tumor defense mechanism.

The angiogenic switch during tumorigenesis is thought to be induced by a change in the balance of pro- angiogenic and anti-angiogenic factors. To elucidate the biological role of the endogenous angiogenesis inhibitor thrombospondin-2 (TSP-2) during multistep carcinogenesis, we subjected TSP-2-deficient and wild-type mice to a chemical skin carcinogenesis regimen. Surprisingly, TSP-2 expression was strongly upregulated in the mesenchymal stroma of wild-type mice throughout the consecutive stages of tumorigenesis whereas the angiogenesis factor, vascular endothelial growth factor, was induced predominantly in tumor cells. TSP-2 deficiency dramatically enhanced susceptibility to skin carcinogenesis and resulted in accelerated and increased tumor formation. The angiogenic switch occurred in early stages of pre-malignant tumor formation, and tumor angiogenesis was significantly enhanced in TSP-2-deficient mice. While TSP-2 deficiency did not affect tumor differentiation or proliferation, tumor cell apoptosis was significantly reduced. These results reveal upregulation of an endogenous angiogenesis inhibitor during multi step tumorigenesis and identify enhanced stromal TSP-2 expression as a novel host anti-tumor defense mechanism.

Thrombospondin 2, a matricellular protein with diverse functions.

Thrombospondin (TSP) 2 is a close relative of TSP1 but differs in its temporal and spatial distribution in the mouse. This difference in expression undoubtedly reflects the marked disparity in the DNA sequences of the promoters in the genes encoding the two proteins. The synthesis of TSP2 occurs primarily in connective tissues of the developing and growing mouse. In the adult animal the protein is again produced in response to tissue injury and in association with the growth of tumors. Despite the abnormalities in collagen fibrillogenesis, fragility of skin, and laxity of tendons and ligaments observed in the TSP2-null mouse, TSP2 does not appear to contribute directly to the structural integrity of connective tissue elements. Instead, emerging evidence supports a mode of action of TSP2 'at a distance', i.e. by modulating the activity and bioavailability of proteases and growth factors in the pericellular environment and, very likely, by interaction with cell-surface receptors. Thus, TSP2 qualifies as a matricellular protein, as defined in the introduction to this minireview series. The phenotype of TSP2-null mice has been very helpful in providing clues to the functions of TSP2. In addition to histological and functional abnormalities in connective tissues, these mice display an increased vascularity of the dermis and subdermal tissues, increased endosteal bone growth, a bleeding defect, and a marked adhesive defect of dermal fibroblasts. Our laboratory has established that TSP2 binds matrix metalloproteinase 2 (MMP2) and that the adhesive defect in TSP2-null fibroblasts results from increased MMP2 activity. The investigation of the basis for the other defects in the TSP2-null mouse is likely to yield equally interesting results.

Matricellular proteins as modulators of cell-matrix interactions: adhesive defect in thrombospondin 2-null fibroblasts is a consequence of increased levels of matrix metalloproteinase-2.

Thrombospondin 2 (TSP2)-null mice, generated by disruption of the Thbs2 gene, display a variety of connective tissue abnormalities, including fragile skin and the presence of abnormally large collagen fibrils with irregular contours in skin and tendon. In this study we demonstrate that TSP2-null skin fibroblasts show a defect in attachment to a number of matrix proteins, and a reduction in cell spreading. To investigate the molecular mechanisms responsible for these abnormal cell-matrix interactions, we compared the levels of matrix metalloproteinases (MMPs) in wild-type and mutant fibroblasts. Isolation and analysis of gelatinases from conditioned media by gelatin-agarose affinity chromatography and gelatinolytic assays demonstrated that TSP2-null fibroblasts produce a 2-fold increase in gelatinase A (MMP2) compared with wild-type cells. The adhesive defect was corrected by treatment of TSP2-null fibroblasts with soluble TSP2, with the MMP inhibitors BB94 and tissue inhibitor of metalloproteinase-2, and with a neutralizing antibody to MMP2. Moreover, stable transfection of TSP2-null fibroblasts with mouse TSP2 cDNA corrected both the adhesive defect and the altered expression of MMP2. Finally, MMP2 was shown to interact with TSP2 in a direct-binding plate assay. We conclude that TSP2 plays an important role in cell-matrix interactions, and that a deficiency in the protein results in increased levels of MMP2 that contribute to the adhesive defect in TSP2-null fibroblasts and could play a role in the complex phenotype of TSP2-null mice.

Increased marrow-derived osteoprogenitor cells and endosteal bone formation in mice lacking thrombospondin 2.

The phenotype of thrombospondin 2 (TSP2)-null mice includes abnormalities in collagen fibrils and increases in ligamentous laxity, vascular density, and bleeding time. In this study, analyses by computerized tomography (CT) revealed that cortical density was increased in long bones of TSP2-null mice. Histomorphometric analysis showed that the mid-diaphyseal endosteal bone formation rate (BFR) of TSP2-null mice was increased in comparison with that of wild-type (WT) animals. Although microgeometric analysis showed that periosteal and endosteal radii were reduced, the mechanical properties of femurs from TSP2-null mice were not significantly different from those of controls, presumably because of the concomitant increase in endosteal bone mass. Bone loss in ovariectomized mice was equivalent for WT and mutant mice, a finding that indicates that TSP2-null animals are capable of normal bone resorption. To further explore the cellular basis for the increased endosteal BFR in TSP2-null mice, marrow stromal cells (MSCs) were isolated and examined in vitro. These cells were found to be present in increased numbers in a colony forming unit (CFU) assay and showed an increased rate of proliferation in vitro. We conclude that TSP2 regulates the proliferation of osteoblast progenitors, directly or indirectly, and that in its absence endosteal bone formation is increased.

Thrombospondin 2 modulates collagen fibrillogenesis and angiogenesis.

Thrombospondin 2 (TSP2)-null mice, generated by targeted disruption of the Thbs2 gene, display a complex phenotype that is characterized, in part, by a variety of connective tissue abnormalities and increased vascular density in skin and subcutaneous tissues. In this paper we summarize the evidence that TSP2 functions as a matricellular protein to influence cell function by modulating cell-matrix interactions, rather than acting as an integral component of the matrix. Thus, the structurally abnormal collagen fibrils detected in skin appear to be the consequence of the defective adhesion demonstrated by dermal fibroblasts in culture that, in turn, result from increased matrix metalloproteinase 2 (MMP2, gelatinase A) production by these cells. Corroborating evidence for such a mode of action comes from transmission electron microscopic images of developing flexor muscle tendons that show distinct abnormalities in fibroblast-collagen fibril interactions in TSP2-null tissue. The increased vascular density seen in skin of TSP2-null mice can be reproduced in a number of models of injury, including subcutaneous implantation of polyvinyl alcohol sponges and silicone rubber discs, and excisional skin wounds. Experiments are proposed to distinguish between a primarily endothelial cell versus an extracellular matrix origin for the increased angiogenesis in TSP2-null mice.

Accelerated wound healing in mice with a disruption of the thrombospondin 2 gene.

Mice that lack the extracellular matrix protein thrombospondin 2 have, among several abnormalities, an increase in vascular density, abnormal collagen fibrils, and dermal fibroblasts that are defective in adhesion. These findings suggested that responses involving these processes, such as wound healing, might be altered. To investigate the healing process, excisional wounds were made with the aid of a biopsy punch. Such wounds, observed over a 14 d period, appeared to heal at an accelerated rate and with less scarring in thrombospondin 2-null mice. Histologic analysis of thrombospondin 2-null wound sites revealed the presence of an irregularly organized and highly vascularized granulation tissue. In addition, thrombospondin 2-null wounds retained a higher total cellular content, than control wounds. No differences in wound re-epithelization rates were observed, but thrombospondin 2-null epithelia formed rete pegs and were thicker than control epithelia. By immunohistochemistry, we detected elevated levels and an irregular deposition pattern for fibronectin in thrombospondin 2-null wounds, observations that correlated with the abnormal collagen organization in the granulation tissue. Immunostaining for thrombospondin 2 in control wounds showed that the protein is present in both early and late wounds, in a scattered cell-associated pattern or widely distributed cell- and matrix-associated pattern, respectively. Our results suggest that thrombospondin 2 plays a crucial part in the organization and vascularization of the granulation tissue during healing, possibly by modulating fibroblast-matrix interactions in early wounds and regulating the extent of angiogenesis in late wounds.

Mice that lack the angiogenesis inhibitor, thrombospondin 2, mount an altered foreign body reaction characterized by increased vascularity.

Disruption of the thrombospondin 2 gene (Thbs2) in mice results in a complex phenotype characterized chiefly by abnormalities in fibroblasts, connective tissues, and blood vessels. Consideration of this phenotype suggested to us that the foreign body reaction (FBR) might be altered in thrombospondin 2 (TSP2)-null mice. To investigate the participation of TSP2 in the FBR, polydimethylsiloxane (PDMS) and oxidized PDMS (ox-PDMS) disks were implanted in TSP2-null and control mice. Growth of TSP2-null and control skin fibroblasts in vitro also was evaluated on both types of disks. Normal fibroblasts grew as a monolayer on both surfaces, but attachment of the cells to ox-PDMS was weak and sensitive to movement. TSP2-null fibroblasts grew as aggregates on both surfaces, and their attachment was further compromised on ox-PDMS. After a 4-week implantation period, both types of PDMS elicited a similar FBR with a collagenous capsule in both TSP2-null and control mice. However, strikingly, the collagenous capsule that formed in TSP2-null mice was highly vascularized and thicker than that formed in normal mice. In addition, abnormally shaped collagen fibers were observed in capsules from mutant mice. These observations indicate that the presence or absence of an extracellular matrix component, TSP2, can influence the nature of the FBR, in particular its vascularity. The expression of TSP2 therefore could represent a molecular target for local inhibitory measures when vascularization of the tissue surrounding an implanted device is desired.

The distribution of the matricellular protein thrombospondin 2 in tissues of embryonic and adult mice.

Mice that lack the matricellular protein thrombospondin 2 (TSP2) develop a pleiotropic phenotype characterized by morphological changes in connective tissues, an increase in vascular density, and a propensity for bleeding. Furthermore, dermal cells derived from TSP2-null mice display adhesion defects, a finding that implicates TSP2 in cell-matrix interactions. To gain a better understanding of the participation of TSP2 in the development and maturation of the mouse, we examined its distribution in embryonic and adult tissues. Special attention was paid to the presence of TSP2 in collagen fibers, because collagen fibrils in the TSP2-null mouse appear to be irregular in size and contour by electron microscopy. Immunohistochemical analysis of Day 15 and Day 18 embryos revealed TSP2 in areas of chondrogenesis, osteogenesis, and vasculogenesis, and in dermal and other connective tissue-forming cells. Distinctly different patterns of deposition of TSP2 were observed in areas of developing cartilage and bone at Days 15 and 18 of embryonic development. A survey of adult tissues revealed TSP2 in dermal fibroblasts, articular chondrocytes, Purkinje cells in the cerebellum, Leidig cells in the testis, and in the adrenal cortex. Dermal fibroblasts were also shown to synthesize TSP2 in vitro. The distribution of TSP2 during development is in keeping with its participation in the formation of a variety of connective tissues. In adult tissues, TSP2 is located in the pericellular environment, where it can potentially influence the cell-matrix interactions associated with cell movement and tissue repair.

Mice that lack thrombospondin 2 display connective tissue abnormalities that are associated with disordered collagen fibrillogenesis, an increased vascular density, and a bleeding diathesis.

Thrombospondin (TSP) 2, and its close relative TSP1, are extracellular proteins whose functions are complex, poorly understood, and controversial. In an attempt to determine the function of TSP2, we disrupted the Thbs2 gene by homologous recombination in embryonic stem cells, and generated TSP2-null mice by blastocyst injection and appropriate breeding of mutant animals. Thbs2-/- mice were produced with the expected Mendelian frequency, appeared overtly normal, and were fertile. However, on closer examination, these mice displayed a wide variety of abnormalities. Collagen fiber patterns in skin were disordered, and abnormally large fibrils with irregular contours were observed by electron microscopy in both skin and tendon. As a functional correlate of these findings, the skin was fragile and had reduced tensile strength, and the tail was unusually flexible. Mutant skin fibroblasts were defective in attachment to a substratum. An increase in total density and in cortical thickness of long bones was documented by histology and quantitative computer tomography. Mutant mice also manifested an abnormal bleeding time, and histologic surveys of mouse tissues, stained with an antibody to von Willebrand factor, showed a significant increase in blood vessels. The basis for the unusual phenotype of the TSP2-null mouse could derive from the structural role that TSP2 might play in collagen fibrillogenesis in skin and tendon. However, it seems likely that some of the diverse manifestations of this genetic disorder result from the ability of TSP2 to modulate the cell surface properties of mesenchymal cells, and thus, to affect cell functions such as adhesion and migration.

Biochemical characterization, developmental expression, and induction of the immune protein scolexin from Manduca sexta.

The immune protein, scolexin, a bacteria-induced, larva-specific protein from Manduca sexta, was shown to exist in the hemolymph in two isoelectric forms designated herein as scolexin-1 and scolexin-2 (native M(r) approximately 72 kd). These two charge isomers appeared to share the same amino acid composition. Scolexin is composed of two subunits (peptide M(r) approximately 36 kd) that possess the same N-terminus. Scolexin-2 was subjected to glycosyl composition analysis, revealing the presence of galactose, glucose, mannose, xylose, and sialic acid residues. Hybridization of epidermal RNA with oligonucleotides deduced from the scolexin N-terminal sequence showed a continuous decline in mRNA following day 0 of the 5th larval instar. By employing in vitro protein labelling, it was found that organ cultures of the epidermis from immune larvae showed a greater ability over that of naive epidermal cultures to synthesize scolexin; these data reflected the inducible response seen in the hemolymph, and confirm other data indicating that the epidermis is an important site of scolexin biosynthesis.

In vivo distribution of immune protein scolexin in bacteria-injected Manduca sexta larvae.

The Manduca sexta larva-specific immune protein, scolexin, was isolated and (14)CH(3)-labelled by reductive alkylation. The influence of the bacterium Streptococcus faecalis on the hemocoelic distribution of the labelled scolexin was then analyzed. During bacterial challenge, most of the scolexin signal was detected in association with the hemocyte aggregations and nodules which formed; in this respect the protein sometimes appeared to be associated with hemocytes which had phagocytized bacteria, while at other times it was most concentrated in the nodule-associated, and free, coagulum. Areas of high scolexin activity were sometimes detected at various sites on the surface of the fat body. The scolexin did not appear to bind directly to bacterial cells. Up to 24 hr following the injection of S. faecalis, the larvae were still carrying out the formation of nodules; unlike the nodules of the 3 and 6 hr intervals, the nodules observed at 21-24 hr were covered with an apparently humorally derived, coagular capsule.