Implanted foreign materials , used to restore or assist tissue function , elicit an initial acute... more Implanted foreign materials , used to restore or assist tissue function , elicit an initial acute inflammatory response followed by chronic fibrosis that leads to the entrapment of the biomaterial in a thick , poorly vascularized collagenous capsule. Matricellular proteins , secreted macromolecules that interact with extracellular matrix proteins but do not in themselves serve structural roles , have been identified as important mediators of the foreign body response that includes inflammation , angiogenesis , and collagen synthesis and assembly. In this report we delineate functions of hevin and SPARC , two homologs of the SPARC family of matricellular proteins , in the foreign body response. Despite their sequence similarity, hevin and SPARC mediate different aspects of this fibrotic response. Using mice with targeted gene deletions , we show that hevin is central to the progression of biomaterial-induced inflammation whereas SPARC regulates the formation of the collagenous capsule. Although vascular density within the capsule is unaltered in the absence of either protein , SPARChevin double-null capsules show substantially increased numbers of vessels , indicating compensatory functions for these two proteins in the inhibition of angiogenesis. These results provide important information for further development of implant technology.
Cell migration is essential to many physiological processes, including angiogenesis, which is cri... more Cell migration is essential to many physiological processes, including angiogenesis, which is critical to the success of implanted biomaterials and tissue-engineered constructs. Gradients play an important role in cell migration. Previous work on cell migration has been mostly executed either in the concentration gradients of stimuli (e.g., VEGF) in bulk or hydrogels or on the surface-density gradients of ECM proteins (e.g., fibronectin) or small ligands (e.g., RGD). Little work has been done to investigate how cell migration responds to the surface-density gradients of growth factors. No work has been done to study how the surface gradients of both adhesive proteins and growth factors influence cell migration. In this work, we studied the effect of the surface-density gradients of fibronectin (FN), VEGF, or both proteins on endothelial cell migration. Gradients with different slopes were prepared to study how the gradient slope affects cell migration. The gradients were generated by first forming a counter-propagating C 15 COOH/C 11 OH selfassembled monolayer (SAM) gradient using a surface electrochemistry approach, followed by activating the -COOH moieties and covalently immobilizing proteins onto the surface. Fourier transform infrared spectra and X-ray photoelectron spectroscopy were used to characterize the SAM and protein gradients, respectively. A free cell migration assay using bovine aortic endothelial cells was performed on various gradient surfaces or on surfaces with uniform protein density. Results showed that cells on the surface-density gradients of FN, VEGF, or both proteins moved faster along the gradient direction than on the respective uniform control surface after 24-h cell culture. It is also shown that for each protein or protein combination, the directional cell displacement was not statistically different between two gradients with different slopes. Results show that the directional cell migration was increased by about 2-fold on the VEGF gradient as compared to the FN gradient and was further increased by another 2-fold on the combined gradients of both proteins as compared to the VEGF gradient alone. This is the first work to create surface-density gradients of VEGF and the first study to generate a combined surface gradient of growth factor and ECM protein to investigate their effect on cell migration on surfaces. This work broadens our understanding of the directional movement of endothelial cells. Our findings provide useful information for directing cell migration into tissue-engineered constructs and can be potentially used for those applications where cell migration is critical, such as angiogenesis.
Journal of Biomaterials Science-polymer Edition, 2008
The foreign body reaction (FBR), which leads to the encapsulation of implanted biomaterials, has ... more The foreign body reaction (FBR), which leads to the encapsulation of implanted biomaterials, has been implicated in the failure of many medical devices. The protein layer that is nonspecifically adsorbed onto the implant surface immediately after implantation is thought to dictate this reaction. It is hypothesized that biomaterial surfaces having specific proteins with precisely controlled orientations will decrease the FBR. Previously, we have reported that osteopontin (OPN) adsorbed on positively charged surfaces has a preferable orientation for in vitro cell adhesion and spreading as compared to negatively charged surfaces. It is expected that coating a layer of OPN in its preferred orientation on an implant surface will decrease the FBR. In this work, in vivo studies were performed to test this hypothesis. A positively charged polymer (p(HEMA-co-AEMA)) and a negatively charged polymer (p(HEMA-co-CEA)) coated with OPN were implanted subcutaneously in wild-type mice for 7 or 28 days. Uncoated polymers were used as control. For the 7-day implants, cells on OPN-coated p(HEMA-co-AEMA) spread more than cells on the other three materials. Following 28 days of implantation the implants were explanted and the capsule thickness and vascularity around the implants were characterized. Additionally, the macrophage and foreign body giant cells (FBGCs) around the implants were quantified. It was found in this study that the modification of the positively charged polymer surface with OPN in a controlled orientation led to a reduction in the foreign body reaction as determined by capsule thickness. Our finding provides valuable information for designing better biocompatible biomaterials with improved in vivo performance.
Vitronectin (Vn) is not only a major adhesive glycoprotein in plasma but also regulates cell-medi... more Vitronectin (Vn) is not only a major adhesive glycoprotein in plasma but also regulates cell-mediated proteolytic enzyme cascades, including the complement, coagulation, and fibrinolytic systems. This broad functional activity suggests that Vn may also play a critical role in development. To begin to investigate this possibility, we studied Vn gene expression during murine embryogenesis. In situ hybridization analysis of embryonic tissues revealed Vn mRNA primarily in the liver and the central nervous system (CNS). In the liver, Vn mRNA was detected by day 10, the level increasing at later developmental stages. In the CNS, Vn mRNA was also detected as early as day 10 and was confined to the floor plate. However, as development proceeded, high levels of Vn transcripts became prominent in the meninges of the cortex and spinal cord, and in close proximity to brain capillaries. The perikarya of most neurons lacked Vn mRNA. Unexpectedly, high levels of Vn mRNA were associated with capillaries of the CNS, but not with blood vessels of peripheral organs. These results indicate that Vn is expressed in a spatially and temporally distinct pattern during murine embryogenesis, and suggest that the Vn transcript may be a CNS-specific vascular marker. o 1995 WiIey-Liss, Inc.
Decorin is known to influence tissue tensile strength and cellular phenotype. Therefore, decorin ... more Decorin is known to influence tissue tensile strength and cellular phenotype. Therefore, decorin is likely to have an impact on tissue repair, including cutaneous wound healing. In this study, cutaneous healing of both excisional and incisional full-thickness dermal wounds was studied in decorin-deficient (Dcn À/À ) animals. A statistically significant delay in excisional wound healing in the Dcn À/À mice occurred at 4 and 10 days postwounding and, in incisional wounds at 4, 10, and 18 days when compared with wild-type (Dcn À/À ) controls. Fibrovascular invasion into polyvinylalcohol sponges was significantly increased by day 18 in Dcn À/À mice relative to Dcn 1/1 mice. The 18-day sponge implants in the Dcn À/À mice showed a marked accumulation of biglycan when compared with the corresponding implants in Dcn 1/1 mice. Thus, regulated production of decorin may serve as an excellent therapeutic approach for modifying impaired wound healing and harmful foreign body reactions.
Journal of Histochemistry and Cytochemistry, Dec 1, 1999
SPARC (secreted protein, acidic and rich in cysteine) is a unique matricellular glycoprotein that... more SPARC (secreted protein, acidic and rich in cysteine) is a unique matricellular glycoprotein that is expressed by many different types of cells and is associated with development, remodeling, cell turnover, and tissue repair. Its principal functions in vitro are counteradhesion and antiproliferation, which proceed via different signaling pathways. SPARC consists of three domains, each of which has independent activity and unique properties. The extracellular calcium binding module and the follistatin-like module have been recently crystallized. Specific interactions between SPARC and growth factors, extracellular matrix proteins, and cell surface proteins contribute to the diverse activities described for SPARC in vivo and in vitro. The location of SPARC in the nuclear matrix of certain proliferating cells, but only in the cytosol of postmitotic neurons, indicates potential functions of SPARC as a nuclear protein, which might be involved in the regulation of cell cycle progression and mitosis. High levels of SPARC have been found in adult eye, and SPARC-null mice exhibit cataracts at 1-2 months of age. This animal model provides an excellent opportunity to confirm and explore some of the properties of SPARC, to investigate cataractogenesis, and to study SPARC-related family proteins, e.g., SC1/hevin, a counteradhesive matricellular protein that might functionally compensate for SPARC in certain tissues.
Secreted protein, acidic and rich in cysteine (SPARC) has been described as a counteradhesive mat... more Secreted protein, acidic and rich in cysteine (SPARC) has been described as a counteradhesive matricellular protein with a diversity of biological functions associated with morphogenesis, remodeling, cellular migration, and proliferation. We have produced mouse SPARC with a FLAG-tag at the N-terminus of SPARC (Flag-SPARC, FSP) in a Bac-to-Bac baculoviral expression system. After affinity purification, this procedure yields SPARC of high purity, with an electrophoretic mobility of ,44 kDa under reducing conditions, and ,38-39 kDa under non-reducing conditions. Unexpectedly, FSP adsorbed to plastic supported cell attachment and spreading, in a calcium-dependent manner. The adhesive activity of native FSP was inhibited by prior incubation with anti-SPARC IgG. Cell adhesion to FSP induced the formation of filopodia and lamellipodia but not focal adhesions that were prominent on cells that were attached to fibronectin. In addition, FSP induced the tyrosine phosphorylation of FAK and paxillin in attached epithelial cells. Erk1/2 and Rac were also activated in cells attached to FSP, but at a lower level in comparison to cells on fibronectin. This study provides new insight into the biological functions of SPARC, a matricellular protein with important roles in cell-extracellualr matrix interactions.
Secreted protein acidic and rich in cysteine (SPARC/osteonectin/BM-40) is a matricellular protein... more Secreted protein acidic and rich in cysteine (SPARC/osteonectin/BM-40) is a matricellular protein that functions in wound healing. Fibrinogen is a plasma protein involved in many aspects of wound healing, such as inflammation, fibrosis and thrombosis. In this study, the binding of SPARC to both native and plasmin-cleaved fibrinogen under physiological conditions was examined by the use of a surface plasmon resonance (SPR) biosensor. We show that SPARC binds to plasmin-cleaved fibrinogen, but not to native fibrinogen. SPARC binds to both fibrinogen fragments D and E fg D and fg E with similar dissociation constants (8.67 x 10(-8) M for Fg D and 1.61 x 10(-7) M for Fg E). Results from endothelial cell proliferation assays show that the binding of SPARC to Fg E suppressed the inhibition of proliferation by SPARC, whereas the binding of SPARC to Fg D did not influence the activity of SPARC on the cell cycle. The interaction of SPARC with fibrinogen fragments D and E, which are produced as a result of proteolytic activation of fibrinolysis, reveals potential storage sites in provisional extracellular matrix for SPARC during the wound healing process and indicates a regulatory role of SPARC in fibrinolysis and angiogenesis.
Transcription of the plasminogen activator inhibitor type-1 (PAI-1) gene appears to be growth sta... more Transcription of the plasminogen activator inhibitor type-1 (PAI-1) gene appears to be growth state regulated in several cell types (e.g. , Ryan and Higgins, 1993, J Cell Physiol 155:376-384; Mu et al., 1998, J Cell Physiol 174:90-98). Transit of serum-stimulated normal rat kidney (NRK) epthelial cells through the first division cycle after release from quiescence (G(0)) provided a model system to assess the kinetics and mechanisms underlying PAI-1 expression in a growth "activated" phenotype. PAI-1 mRNA transcripts increased by more than 20-fold during the G(0)-->G(1) transition; induced expression had immediate-early response characteristics and abruptly declined prior to the onset of DNA synthesis. Transcriptional activity of the PAI-1 gene paralleled the steady-state mRNA abundance profile during this first synchronized growth cycle after release from quiescence. Although PAI-1 mRNA levels were up-regulated (approximately threefold) upon exposure to several different growth factors, neutralizing antibodies to transforming growth factor-beta1 (TGF-beta1) effectively attenuated the more than ninefold serum-associated PAI-1 inductive response by more than 70% (at both the mRNA transcript and protein levels). Similar to the metabolic requirements for serum-mediated PAI-1 transcription, PAI-1 induction upon addition of TGF-beta1 to quiescent NRK cell cultures was actinomycin D sensitive and resistant to cyclohexamide and puromycin, suggesting a primary mode of transcript control. The response to protein synthesis inhibitors, however, was complex. While cyclohexamide appeared to stabilize, or at least maintain, fetal bovine serum (FBS)- or TGF-beta1-stimulated PAI-1 mRNA levels, puromycin had no such affect. The amplitude and duration of induced PAI-1 expression were the same in either the presence or absence of puromycin. Cyclohexamide when used alone (i.e., in non-FBS- or TGF-beta1-treated cultures), moreover, effectively stimulated PAI-1 induction whereas puromycin was ineffective. Although TGF-beta1 was not a complete mitogen in the NRK cell system, incubation of quiescent renal cell cultures with TGF-beta1, prior to serum stimulation, resulted in a 10- to 12-fold increase in PAI-1 expression coincident with exit out of G(0). These data support a model in which PAI-1 gene expression is closely associated with creation of the growth-activated state and that cell cycle controls appear to be superimposed on the time course of the serum-induced expression of the PAI-1 gene.
Journal of Histochemistry and Cytochemistry, Jun 1, 2004
S U M M A R Y SPARC (osteonectin, BM-40) is a matricellular glycoprotein that is expressed in man... more S U M M A R Y SPARC (osteonectin, BM-40) is a matricellular glycoprotein that is expressed in many embryogenic and adult tissues undergoing remodeling or repair. SPARC modulates cellular interaction with the extracellular matrix (ECM), inhibits cell adhesion and proliferation, and regulates growth factor activity. To explore further the function and activity of this protein in tissue homeostasis, we have developed several monoclonal antibodies (MAbs) that recognize distinct epitopes on SPARC. The MAbs bind to SPARC with high affinity and identify SPARC by ELISA, Western blotting, immunoprecipitation, immunocytochemistry, and/or immunohistochemistry. The MAbs were also characterized in functional assays for potential alteration of SPARC activity. SPARC binds to collagen I and laminin-1 through an epitope defined by MAb 293; this epitope is not involved in the binding of SPARC to collagen III. The other MAbs did not interfere with the binding of SPARC to collagen I or III or laminin-1. Inhibition of the anti-adhesive effect of SPARC on endothelial cells by MAb 236 was also observed. Functional analysis of SPARC in the presence of these novel MAbs now confirms that the activities ascribed to this matricellular protein can be assigned to discrete subdomains. (J Histochem Cytochem 52: [723][724][725][726][727][728][729][730][731][732][733] 2004)
Purpose. This investigation was undertaken to determine whether the matricellular protein SPARC i... more Purpose. This investigation was undertaken to determine whether the matricellular protein SPARC is expressed in the human trabecular meshwork (TM) and cultured human trabecular meshwork cells. Methods. Human donor trabecular meshwork and cultured cells obtained from trabecular meshwork were used in this study. Total RNA was obtained from TM and cultured TM endothelial cells, and RT-PCR was done with primers specific for SPARC. Western blotting was performed on donor TMs using an anti-SPARC monoclonal antibody prepared against rHuSPARC. Confocal microscopy was used to determine the distribution of SPARC in human anterior segments, and immunofluorescence on cultured TM cells was performed with the anti-SPARC antibody. Results. SPARC mRNA was expressed both in TM and in cultured TM cells. Immunoblotting for SPARC showed a doublet with a molecular mass ,43 kDa. The ratio of the doublet bands varied with each of the samples; some of the cultured cells and the tissue samples exhibited more of the upper band, and other cultured cells contained almost equal amounts of the two bands. The upper band was shown to be a glycosylated form of SPARC. Immunofluorescence showed that SPARC was expressed in the cultured TM, and confocal microscopy with the anti-SPARC antibody demonstrated the presence of this protein in the TM and in other tissues in the anterior segment. Conclusions. Our data conclusively show that SPARC mRNA and protein are present in non-glaucomatous TM tissue and in cultured TM cells. Because of its effect on matrix metalloproteinases, SPARC may play a role in the regulation of intraocular pressure.
Secreted Protein Acidic and Rich in Cystein (SPARC)/osteonectin is a nonstructural matricellular ... more Secreted Protein Acidic and Rich in Cystein (SPARC)/osteonectin is a nonstructural matricellular protein involved in cell-matrix interaction during tissue remodeling and embryonic development. Using a novel monoclonal antibody (10-255), we examined immunohistochemically the patterns of SPARC expression in non-small cell lung cancer (NSCLC). High levels of SPARC in normal lung were confined exclusively to the bronchial cartilage. In NSCLC tissues, cancer cells were unreactive in 107 of 113 cases analyzed (95%), whereas substantial production of SPARC by stromal fibroblasts was noted in 42 of 113 cases (37%). Stromal SPARC was linked with tumor necrosis (P = 0.01) and, marginally, with node metastasis (P = 0.07), as well as with high levels of carbonic anhydrase 9 and LDH in cancer cells (P = 0.0001 and P = 0.01, respectively). SPARC was also coincident with enhanced levels of cancer cell differentiated embryo-chondrocyte expressed gene 1, hypoxia inducible factor 2alpha, and thymidine phosphorylase (P = 0.01, P = 0.05, and P = 0.03, respectively). Although endothelial reactivity for SPARC was noted only in small, immature vessels, SPARC production by stroma cells supported a high degree of vascular maturation (indicated by the presence of subendothelial lamina lucida). Survival analysis revealed a significant association of stromal SPARC with poor prognosis (P = 0.006), a finding that was also confirmed in multivariate models. In NSCLC, SPARC is selectively synthesized by the cells of the tumoral stroma. The strong association of this feature with markers of intratumoral hypoxia and acidity indicates an interesting link between cancer cell metabolism and the induction of a supportive stroma that favors cancer cell invasion and migration that lead to an ominous clinical outcome.
Annals of the New York Academy of Sciences, Feb 1, 1990
All molecular weights were based on comparative migration with globular protein standards. ' No i... more All molecular weights were based on comparative migration with globular protein standards. ' No immunoreactive proteins were detected.
SPARC (secreted protein, acidic and rich in cysteine) is a matricellular glycoprotein that regula... more SPARC (secreted protein, acidic and rich in cysteine) is a matricellular glycoprotein that regulates morphogenesis, cellular proliferation, and differentiation. SPARC is a critical factor in the development and maintenance of lens transparency in mice. SPARC-null mice develop lenticular opacity at an early age that progresses gradually to mature cataract. Despite the high level of homology between the mouse and human genes, little is known about SPARC in the human lens. We have studied the expression of SPARC protein in human lens and surrounding ocular tissues from normal human donors (60±70 years old). Immunohistochemical and immunoblot analyses were conducted on lens, aqueous humor, vitreous, ciliary epithelium, pigment epithelium, cornea and retina. The epithelia and capsule of the lens contained SPARC, whereas the cortical and nuclear ®bers did not. In contrast, the aqueous humor and vitreous, which provide nutrients to the lens and regulate its development and function, contained signi®cant amounts of SPARC. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracts of various ocular tissues revealed bands of 43 and 29 kD after disul®de bond reduction that were reactive with anti-SPARC IgG. Despite the presence of protease inhibitors during sample preparation, we observed cleavage of intact SPARC to a 29 kD fragment, a peptide reported in other tissues and attributed to endogenous proteolysis. In addition, bands of molecular mass 150 and 200 kD were present that appeared to be disul®de-bonded complexes of SPARC monomers. Human cornea, ciliary epithelium, pigment epithelium and retina also contained SPARC. The presence of SPARC in the aqueous humor and vitreous, as well as in the lens, indicates a functional importance of SPARC in adult human eye as well as in lens development.
Utilizing subcutaneous tumor models, we previously validated SPARC (secreted protein acidic and r... more Utilizing subcutaneous tumor models, we previously validated SPARC (secreted protein acidic and rich in cysteine) as a key component of the stromal response, where it regulated tumor size, angiogenesis and extracellular matrix deposition. In the present study, we demonstrate that pancreatic tumors grown orthotopically in Sparc-null (Sparc -/-) mice are more metastatic than tumors grown in wild-type (Sparc +/+ ) littermates. Tumors grown in Sparc -/-mice display reduced deposition of fibrillar collagens I and III, basement membrane collagen IV and the collagen-associated proteoglycan decorin. In addition, microvessel density and pericyte recruitment are reduced in tumors grown in the absence of host SPARC. However, tumors from Sparc -/-mice display increased permeability and perfusion, and a subsequent decrease in hypoxia. Finally, we found that tumors grown in the absence of host SPARC exhibit an increase in alternatively activated macrophages. These results suggest that increased tumor burden in the absence of host SPARC is a consequence of reduced collagen deposition, a disrupted vascular basement membrane, enhanced vascular function and an immune-tolerant, pro-metastatic microenvironment.
Implanted foreign materials , used to restore or assist tissue function , elicit an initial acute... more Implanted foreign materials , used to restore or assist tissue function , elicit an initial acute inflammatory response followed by chronic fibrosis that leads to the entrapment of the biomaterial in a thick , poorly vascularized collagenous capsule. Matricellular proteins , secreted macromolecules that interact with extracellular matrix proteins but do not in themselves serve structural roles , have been identified as important mediators of the foreign body response that includes inflammation , angiogenesis , and collagen synthesis and assembly. In this report we delineate functions of hevin and SPARC , two homologs of the SPARC family of matricellular proteins , in the foreign body response. Despite their sequence similarity, hevin and SPARC mediate different aspects of this fibrotic response. Using mice with targeted gene deletions , we show that hevin is central to the progression of biomaterial-induced inflammation whereas SPARC regulates the formation of the collagenous capsule. Although vascular density within the capsule is unaltered in the absence of either protein , SPARChevin double-null capsules show substantially increased numbers of vessels , indicating compensatory functions for these two proteins in the inhibition of angiogenesis. These results provide important information for further development of implant technology.
Cell migration is essential to many physiological processes, including angiogenesis, which is cri... more Cell migration is essential to many physiological processes, including angiogenesis, which is critical to the success of implanted biomaterials and tissue-engineered constructs. Gradients play an important role in cell migration. Previous work on cell migration has been mostly executed either in the concentration gradients of stimuli (e.g., VEGF) in bulk or hydrogels or on the surface-density gradients of ECM proteins (e.g., fibronectin) or small ligands (e.g., RGD). Little work has been done to investigate how cell migration responds to the surface-density gradients of growth factors. No work has been done to study how the surface gradients of both adhesive proteins and growth factors influence cell migration. In this work, we studied the effect of the surface-density gradients of fibronectin (FN), VEGF, or both proteins on endothelial cell migration. Gradients with different slopes were prepared to study how the gradient slope affects cell migration. The gradients were generated by first forming a counter-propagating C 15 COOH/C 11 OH selfassembled monolayer (SAM) gradient using a surface electrochemistry approach, followed by activating the -COOH moieties and covalently immobilizing proteins onto the surface. Fourier transform infrared spectra and X-ray photoelectron spectroscopy were used to characterize the SAM and protein gradients, respectively. A free cell migration assay using bovine aortic endothelial cells was performed on various gradient surfaces or on surfaces with uniform protein density. Results showed that cells on the surface-density gradients of FN, VEGF, or both proteins moved faster along the gradient direction than on the respective uniform control surface after 24-h cell culture. It is also shown that for each protein or protein combination, the directional cell displacement was not statistically different between two gradients with different slopes. Results show that the directional cell migration was increased by about 2-fold on the VEGF gradient as compared to the FN gradient and was further increased by another 2-fold on the combined gradients of both proteins as compared to the VEGF gradient alone. This is the first work to create surface-density gradients of VEGF and the first study to generate a combined surface gradient of growth factor and ECM protein to investigate their effect on cell migration on surfaces. This work broadens our understanding of the directional movement of endothelial cells. Our findings provide useful information for directing cell migration into tissue-engineered constructs and can be potentially used for those applications where cell migration is critical, such as angiogenesis.
Journal of Biomaterials Science-polymer Edition, 2008
The foreign body reaction (FBR), which leads to the encapsulation of implanted biomaterials, has ... more The foreign body reaction (FBR), which leads to the encapsulation of implanted biomaterials, has been implicated in the failure of many medical devices. The protein layer that is nonspecifically adsorbed onto the implant surface immediately after implantation is thought to dictate this reaction. It is hypothesized that biomaterial surfaces having specific proteins with precisely controlled orientations will decrease the FBR. Previously, we have reported that osteopontin (OPN) adsorbed on positively charged surfaces has a preferable orientation for in vitro cell adhesion and spreading as compared to negatively charged surfaces. It is expected that coating a layer of OPN in its preferred orientation on an implant surface will decrease the FBR. In this work, in vivo studies were performed to test this hypothesis. A positively charged polymer (p(HEMA-co-AEMA)) and a negatively charged polymer (p(HEMA-co-CEA)) coated with OPN were implanted subcutaneously in wild-type mice for 7 or 28 days. Uncoated polymers were used as control. For the 7-day implants, cells on OPN-coated p(HEMA-co-AEMA) spread more than cells on the other three materials. Following 28 days of implantation the implants were explanted and the capsule thickness and vascularity around the implants were characterized. Additionally, the macrophage and foreign body giant cells (FBGCs) around the implants were quantified. It was found in this study that the modification of the positively charged polymer surface with OPN in a controlled orientation led to a reduction in the foreign body reaction as determined by capsule thickness. Our finding provides valuable information for designing better biocompatible biomaterials with improved in vivo performance.
Vitronectin (Vn) is not only a major adhesive glycoprotein in plasma but also regulates cell-medi... more Vitronectin (Vn) is not only a major adhesive glycoprotein in plasma but also regulates cell-mediated proteolytic enzyme cascades, including the complement, coagulation, and fibrinolytic systems. This broad functional activity suggests that Vn may also play a critical role in development. To begin to investigate this possibility, we studied Vn gene expression during murine embryogenesis. In situ hybridization analysis of embryonic tissues revealed Vn mRNA primarily in the liver and the central nervous system (CNS). In the liver, Vn mRNA was detected by day 10, the level increasing at later developmental stages. In the CNS, Vn mRNA was also detected as early as day 10 and was confined to the floor plate. However, as development proceeded, high levels of Vn transcripts became prominent in the meninges of the cortex and spinal cord, and in close proximity to brain capillaries. The perikarya of most neurons lacked Vn mRNA. Unexpectedly, high levels of Vn mRNA were associated with capillaries of the CNS, but not with blood vessels of peripheral organs. These results indicate that Vn is expressed in a spatially and temporally distinct pattern during murine embryogenesis, and suggest that the Vn transcript may be a CNS-specific vascular marker. o 1995 WiIey-Liss, Inc.
Decorin is known to influence tissue tensile strength and cellular phenotype. Therefore, decorin ... more Decorin is known to influence tissue tensile strength and cellular phenotype. Therefore, decorin is likely to have an impact on tissue repair, including cutaneous wound healing. In this study, cutaneous healing of both excisional and incisional full-thickness dermal wounds was studied in decorin-deficient (Dcn À/À ) animals. A statistically significant delay in excisional wound healing in the Dcn À/À mice occurred at 4 and 10 days postwounding and, in incisional wounds at 4, 10, and 18 days when compared with wild-type (Dcn À/À ) controls. Fibrovascular invasion into polyvinylalcohol sponges was significantly increased by day 18 in Dcn À/À mice relative to Dcn 1/1 mice. The 18-day sponge implants in the Dcn À/À mice showed a marked accumulation of biglycan when compared with the corresponding implants in Dcn 1/1 mice. Thus, regulated production of decorin may serve as an excellent therapeutic approach for modifying impaired wound healing and harmful foreign body reactions.
Journal of Histochemistry and Cytochemistry, Dec 1, 1999
SPARC (secreted protein, acidic and rich in cysteine) is a unique matricellular glycoprotein that... more SPARC (secreted protein, acidic and rich in cysteine) is a unique matricellular glycoprotein that is expressed by many different types of cells and is associated with development, remodeling, cell turnover, and tissue repair. Its principal functions in vitro are counteradhesion and antiproliferation, which proceed via different signaling pathways. SPARC consists of three domains, each of which has independent activity and unique properties. The extracellular calcium binding module and the follistatin-like module have been recently crystallized. Specific interactions between SPARC and growth factors, extracellular matrix proteins, and cell surface proteins contribute to the diverse activities described for SPARC in vivo and in vitro. The location of SPARC in the nuclear matrix of certain proliferating cells, but only in the cytosol of postmitotic neurons, indicates potential functions of SPARC as a nuclear protein, which might be involved in the regulation of cell cycle progression and mitosis. High levels of SPARC have been found in adult eye, and SPARC-null mice exhibit cataracts at 1-2 months of age. This animal model provides an excellent opportunity to confirm and explore some of the properties of SPARC, to investigate cataractogenesis, and to study SPARC-related family proteins, e.g., SC1/hevin, a counteradhesive matricellular protein that might functionally compensate for SPARC in certain tissues.
Secreted protein, acidic and rich in cysteine (SPARC) has been described as a counteradhesive mat... more Secreted protein, acidic and rich in cysteine (SPARC) has been described as a counteradhesive matricellular protein with a diversity of biological functions associated with morphogenesis, remodeling, cellular migration, and proliferation. We have produced mouse SPARC with a FLAG-tag at the N-terminus of SPARC (Flag-SPARC, FSP) in a Bac-to-Bac baculoviral expression system. After affinity purification, this procedure yields SPARC of high purity, with an electrophoretic mobility of ,44 kDa under reducing conditions, and ,38-39 kDa under non-reducing conditions. Unexpectedly, FSP adsorbed to plastic supported cell attachment and spreading, in a calcium-dependent manner. The adhesive activity of native FSP was inhibited by prior incubation with anti-SPARC IgG. Cell adhesion to FSP induced the formation of filopodia and lamellipodia but not focal adhesions that were prominent on cells that were attached to fibronectin. In addition, FSP induced the tyrosine phosphorylation of FAK and paxillin in attached epithelial cells. Erk1/2 and Rac were also activated in cells attached to FSP, but at a lower level in comparison to cells on fibronectin. This study provides new insight into the biological functions of SPARC, a matricellular protein with important roles in cell-extracellualr matrix interactions.
Secreted protein acidic and rich in cysteine (SPARC/osteonectin/BM-40) is a matricellular protein... more Secreted protein acidic and rich in cysteine (SPARC/osteonectin/BM-40) is a matricellular protein that functions in wound healing. Fibrinogen is a plasma protein involved in many aspects of wound healing, such as inflammation, fibrosis and thrombosis. In this study, the binding of SPARC to both native and plasmin-cleaved fibrinogen under physiological conditions was examined by the use of a surface plasmon resonance (SPR) biosensor. We show that SPARC binds to plasmin-cleaved fibrinogen, but not to native fibrinogen. SPARC binds to both fibrinogen fragments D and E fg D and fg E with similar dissociation constants (8.67 x 10(-8) M for Fg D and 1.61 x 10(-7) M for Fg E). Results from endothelial cell proliferation assays show that the binding of SPARC to Fg E suppressed the inhibition of proliferation by SPARC, whereas the binding of SPARC to Fg D did not influence the activity of SPARC on the cell cycle. The interaction of SPARC with fibrinogen fragments D and E, which are produced as a result of proteolytic activation of fibrinolysis, reveals potential storage sites in provisional extracellular matrix for SPARC during the wound healing process and indicates a regulatory role of SPARC in fibrinolysis and angiogenesis.
Transcription of the plasminogen activator inhibitor type-1 (PAI-1) gene appears to be growth sta... more Transcription of the plasminogen activator inhibitor type-1 (PAI-1) gene appears to be growth state regulated in several cell types (e.g. , Ryan and Higgins, 1993, J Cell Physiol 155:376-384; Mu et al., 1998, J Cell Physiol 174:90-98). Transit of serum-stimulated normal rat kidney (NRK) epthelial cells through the first division cycle after release from quiescence (G(0)) provided a model system to assess the kinetics and mechanisms underlying PAI-1 expression in a growth "activated" phenotype. PAI-1 mRNA transcripts increased by more than 20-fold during the G(0)-->G(1) transition; induced expression had immediate-early response characteristics and abruptly declined prior to the onset of DNA synthesis. Transcriptional activity of the PAI-1 gene paralleled the steady-state mRNA abundance profile during this first synchronized growth cycle after release from quiescence. Although PAI-1 mRNA levels were up-regulated (approximately threefold) upon exposure to several different growth factors, neutralizing antibodies to transforming growth factor-beta1 (TGF-beta1) effectively attenuated the more than ninefold serum-associated PAI-1 inductive response by more than 70% (at both the mRNA transcript and protein levels). Similar to the metabolic requirements for serum-mediated PAI-1 transcription, PAI-1 induction upon addition of TGF-beta1 to quiescent NRK cell cultures was actinomycin D sensitive and resistant to cyclohexamide and puromycin, suggesting a primary mode of transcript control. The response to protein synthesis inhibitors, however, was complex. While cyclohexamide appeared to stabilize, or at least maintain, fetal bovine serum (FBS)- or TGF-beta1-stimulated PAI-1 mRNA levels, puromycin had no such affect. The amplitude and duration of induced PAI-1 expression were the same in either the presence or absence of puromycin. Cyclohexamide when used alone (i.e., in non-FBS- or TGF-beta1-treated cultures), moreover, effectively stimulated PAI-1 induction whereas puromycin was ineffective. Although TGF-beta1 was not a complete mitogen in the NRK cell system, incubation of quiescent renal cell cultures with TGF-beta1, prior to serum stimulation, resulted in a 10- to 12-fold increase in PAI-1 expression coincident with exit out of G(0). These data support a model in which PAI-1 gene expression is closely associated with creation of the growth-activated state and that cell cycle controls appear to be superimposed on the time course of the serum-induced expression of the PAI-1 gene.
Journal of Histochemistry and Cytochemistry, Jun 1, 2004
S U M M A R Y SPARC (osteonectin, BM-40) is a matricellular glycoprotein that is expressed in man... more S U M M A R Y SPARC (osteonectin, BM-40) is a matricellular glycoprotein that is expressed in many embryogenic and adult tissues undergoing remodeling or repair. SPARC modulates cellular interaction with the extracellular matrix (ECM), inhibits cell adhesion and proliferation, and regulates growth factor activity. To explore further the function and activity of this protein in tissue homeostasis, we have developed several monoclonal antibodies (MAbs) that recognize distinct epitopes on SPARC. The MAbs bind to SPARC with high affinity and identify SPARC by ELISA, Western blotting, immunoprecipitation, immunocytochemistry, and/or immunohistochemistry. The MAbs were also characterized in functional assays for potential alteration of SPARC activity. SPARC binds to collagen I and laminin-1 through an epitope defined by MAb 293; this epitope is not involved in the binding of SPARC to collagen III. The other MAbs did not interfere with the binding of SPARC to collagen I or III or laminin-1. Inhibition of the anti-adhesive effect of SPARC on endothelial cells by MAb 236 was also observed. Functional analysis of SPARC in the presence of these novel MAbs now confirms that the activities ascribed to this matricellular protein can be assigned to discrete subdomains. (J Histochem Cytochem 52: [723][724][725][726][727][728][729][730][731][732][733] 2004)
Purpose. This investigation was undertaken to determine whether the matricellular protein SPARC i... more Purpose. This investigation was undertaken to determine whether the matricellular protein SPARC is expressed in the human trabecular meshwork (TM) and cultured human trabecular meshwork cells. Methods. Human donor trabecular meshwork and cultured cells obtained from trabecular meshwork were used in this study. Total RNA was obtained from TM and cultured TM endothelial cells, and RT-PCR was done with primers specific for SPARC. Western blotting was performed on donor TMs using an anti-SPARC monoclonal antibody prepared against rHuSPARC. Confocal microscopy was used to determine the distribution of SPARC in human anterior segments, and immunofluorescence on cultured TM cells was performed with the anti-SPARC antibody. Results. SPARC mRNA was expressed both in TM and in cultured TM cells. Immunoblotting for SPARC showed a doublet with a molecular mass ,43 kDa. The ratio of the doublet bands varied with each of the samples; some of the cultured cells and the tissue samples exhibited more of the upper band, and other cultured cells contained almost equal amounts of the two bands. The upper band was shown to be a glycosylated form of SPARC. Immunofluorescence showed that SPARC was expressed in the cultured TM, and confocal microscopy with the anti-SPARC antibody demonstrated the presence of this protein in the TM and in other tissues in the anterior segment. Conclusions. Our data conclusively show that SPARC mRNA and protein are present in non-glaucomatous TM tissue and in cultured TM cells. Because of its effect on matrix metalloproteinases, SPARC may play a role in the regulation of intraocular pressure.
Secreted Protein Acidic and Rich in Cystein (SPARC)/osteonectin is a nonstructural matricellular ... more Secreted Protein Acidic and Rich in Cystein (SPARC)/osteonectin is a nonstructural matricellular protein involved in cell-matrix interaction during tissue remodeling and embryonic development. Using a novel monoclonal antibody (10-255), we examined immunohistochemically the patterns of SPARC expression in non-small cell lung cancer (NSCLC). High levels of SPARC in normal lung were confined exclusively to the bronchial cartilage. In NSCLC tissues, cancer cells were unreactive in 107 of 113 cases analyzed (95%), whereas substantial production of SPARC by stromal fibroblasts was noted in 42 of 113 cases (37%). Stromal SPARC was linked with tumor necrosis (P = 0.01) and, marginally, with node metastasis (P = 0.07), as well as with high levels of carbonic anhydrase 9 and LDH in cancer cells (P = 0.0001 and P = 0.01, respectively). SPARC was also coincident with enhanced levels of cancer cell differentiated embryo-chondrocyte expressed gene 1, hypoxia inducible factor 2alpha, and thymidine phosphorylase (P = 0.01, P = 0.05, and P = 0.03, respectively). Although endothelial reactivity for SPARC was noted only in small, immature vessels, SPARC production by stroma cells supported a high degree of vascular maturation (indicated by the presence of subendothelial lamina lucida). Survival analysis revealed a significant association of stromal SPARC with poor prognosis (P = 0.006), a finding that was also confirmed in multivariate models. In NSCLC, SPARC is selectively synthesized by the cells of the tumoral stroma. The strong association of this feature with markers of intratumoral hypoxia and acidity indicates an interesting link between cancer cell metabolism and the induction of a supportive stroma that favors cancer cell invasion and migration that lead to an ominous clinical outcome.
Annals of the New York Academy of Sciences, Feb 1, 1990
All molecular weights were based on comparative migration with globular protein standards. ' No i... more All molecular weights were based on comparative migration with globular protein standards. ' No immunoreactive proteins were detected.
SPARC (secreted protein, acidic and rich in cysteine) is a matricellular glycoprotein that regula... more SPARC (secreted protein, acidic and rich in cysteine) is a matricellular glycoprotein that regulates morphogenesis, cellular proliferation, and differentiation. SPARC is a critical factor in the development and maintenance of lens transparency in mice. SPARC-null mice develop lenticular opacity at an early age that progresses gradually to mature cataract. Despite the high level of homology between the mouse and human genes, little is known about SPARC in the human lens. We have studied the expression of SPARC protein in human lens and surrounding ocular tissues from normal human donors (60±70 years old). Immunohistochemical and immunoblot analyses were conducted on lens, aqueous humor, vitreous, ciliary epithelium, pigment epithelium, cornea and retina. The epithelia and capsule of the lens contained SPARC, whereas the cortical and nuclear ®bers did not. In contrast, the aqueous humor and vitreous, which provide nutrients to the lens and regulate its development and function, contained signi®cant amounts of SPARC. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracts of various ocular tissues revealed bands of 43 and 29 kD after disul®de bond reduction that were reactive with anti-SPARC IgG. Despite the presence of protease inhibitors during sample preparation, we observed cleavage of intact SPARC to a 29 kD fragment, a peptide reported in other tissues and attributed to endogenous proteolysis. In addition, bands of molecular mass 150 and 200 kD were present that appeared to be disul®de-bonded complexes of SPARC monomers. Human cornea, ciliary epithelium, pigment epithelium and retina also contained SPARC. The presence of SPARC in the aqueous humor and vitreous, as well as in the lens, indicates a functional importance of SPARC in adult human eye as well as in lens development.
Utilizing subcutaneous tumor models, we previously validated SPARC (secreted protein acidic and r... more Utilizing subcutaneous tumor models, we previously validated SPARC (secreted protein acidic and rich in cysteine) as a key component of the stromal response, where it regulated tumor size, angiogenesis and extracellular matrix deposition. In the present study, we demonstrate that pancreatic tumors grown orthotopically in Sparc-null (Sparc -/-) mice are more metastatic than tumors grown in wild-type (Sparc +/+ ) littermates. Tumors grown in Sparc -/-mice display reduced deposition of fibrillar collagens I and III, basement membrane collagen IV and the collagen-associated proteoglycan decorin. In addition, microvessel density and pericyte recruitment are reduced in tumors grown in the absence of host SPARC. However, tumors from Sparc -/-mice display increased permeability and perfusion, and a subsequent decrease in hypoxia. Finally, we found that tumors grown in the absence of host SPARC exhibit an increase in alternatively activated macrophages. These results suggest that increased tumor burden in the absence of host SPARC is a consequence of reduced collagen deposition, a disrupted vascular basement membrane, enhanced vascular function and an immune-tolerant, pro-metastatic microenvironment.
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Papers by Helene Sage