Downregulation of SPARC Is Associated with Epithelial-Mesenchymal Transition and Low Differentiation State of Biliary Tract Cancer Cells.

BACKGROUND: Biliary tract cancers (BTCs) have a poor prognosis. BTCs are characterized by a prominent desmoplastic reaction which possibly contributes to the aggressive phenotype of this tumor. The desmoplastic reaction includes excessive production and deposition of extracellular matrix proteins such as periostin, secreted protein acidic and rich in cysteine (SPARC), thrombospondin-1, as well as accumulation of α-smooth muscle actin-positive cancer-associated fibroblasts and immune cells, secreting growth factors and cytokines including transforming growth factor (TGF)-ß. In the present study, we investigated the expression of SPARC in BTC as well as its possible regulation by TGF-ß. METHODS: Expression levels of Sparc, TGF-ß1 and its receptor ALK5 were evaluated by quantitative real-time PCR in 6 biliary tract cell lines as well as 1 immortalized cholangiocyte cell line (MMNK-1). RNAs from tumor samples of 7 biliary tract cancer patients were analyzed for expression of Sparc, TGF-ß type II receptor (TbRII) as well as Twist and ZO-1. MMNK-1 cells were stimulated with TGF-ß for 24 h, and Sparc, ZO-1 and E-Cadherin expressions were determined. The presence of SPARC protein was analyzed by immunohistochemistry in tumor specimens from 10 patients. RESULTS: When comparing basal Sparc transcript levels in diverse BTC cell lines to MMNK-1 cells, we found that it was strongly downregulated in all cancer cell lines. The remaining expression levels were higher in highly differentiated cell lines (CCSW1, MZChA1, MZChA2 and TFK-1) than in less differentiated and undifferentiated ones (BDC, SKChA1). Expression of Sparc in BTC patient samples showed a significant positive correlation with expression of the epithelial marker ZO-1. In contrast, the mesenchymal marker Twist and the TbRII showed a trend of negative correlation with expression of Sparc in these samples. TGF-ß exposure significantly downregulated Sparc expression in MMNK-1 cholangiocytes in vitro in parallel to downregulation of epithelial markers (E-Cadherin and ZO-1). Finally, SPARC immunostaining was performed in 10 patient samples, and the correlation between absence of SPARC and survival times was analyzed. CONCLUSIONS: These data imply that a decrease in SPARC expression is correlated with dedifferentiation of BTC cells resulting in enhanced EMT being possibly mediated by TGF-ß. Thereby SPARC levels might be a marker for individual prognosis of a patient, and strategies aiming at inhibition of SPARC downregulation might have potential for new future therapies.

Clinical significance of SPARC in esophageal squamous cell carcinoma.

BACKGROUND: Secreted protein acidic and rich in cysteine (SPARC) is matricellular protein that modulates interactions between cells and the extracellular matrix. The role of SPARC in carcinogenesis is controversialin that SPARC can be a tumor suppressor, but overexpression of SPARC is associated with poorer prognosis. METHODS: We collected 145 esophageal squamous cell carcinoma and adjacent normal tissues in Shantou, a high incidence region for esophageal cancer. The mRNA and protein expression levels of SPARC in cancer tissue and in adjacent normal mucosa were measured by qRT-PCR, western blot and immunohistochemistry (IHC). RESULTS: The mRNA and protein levels of SPARCwere5.78-fold higher in cancer tissues compared with the case-matched normal epithelium. High expression levels of SPARC in ESCC parenchyma, as detected by IHC, were related to lymph node metastasis and poor prognosis (p = 0.049 and p = 0.04). CONCLUSION: High expression of SPARC in the parenchyma may be a potential predictor of prognosis, suggesting SPARC could serve as a therapeutic target in ESCC.

The relationship between SPARC expression in primary tumor and metastatic lymph node of resected pancreatic cancer patients and patients' survival.

BACKGROUND: Previous researches in pancreatic cancer demonstrated a negative correlation between secreted protein acidic and rich in cysteine (SPARC) expression in primary tumor and survival, but not for SPARC expression in lymph node. In the present study, we aimed to evaluate the SPARC expression in various types of tissues and its impact on patients' prognosis. METHODS: The expression of SPARC was examined by immunohistochemistry in resected pancreatic cancer specimens. Kaplan-Meier analyses and Cox proportional hazards regression were applied to assess the mortality risk. RESULTS: A total of 222 tissue samples from 73 patients were collected to evaluate the SPARC expression, which included 73 paired primary tumor and adjacent normal tissues, 38 paired metastatic and normal lymph nodes. The proportion of positive SPARC expression in metastatic lymph node was high (32/38), whereas in normal lymph node it was negative (0/38). Positive SPARC expression in primary tumor cells was associated with a significantly decreased overall survival (P=0.007) and disease-free survival (P=0.003), whereas in other types of tissues it did not show a predictive role for prognosis. Univariate and multivariate analyses both confirmed this significance. CONCLUSION: SPARC can serve a dual function role as both predictor for prognosis and potentially biomarker for lymph node metastasis in resected pancreatic cancer patients.

SPARC expression in patients with high-risk localized soft tissue sarcoma treated on a randomized phase II trial of neo/adjuvant chemotherapy.

BACKGROUND: Treatment for localized soft tissue sarcoma includes surgery and radiation, while the role of chemotherapy is controversial. Biomarkers that could predict therapeutic response or prognosticate overall survival (OS) are needed to define patients most likely to benefit from systemic treatment. Serum protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein that has been evaluated as a potential biomarker in numerous malignancies given its involvement in cell adhesion, proliferation, migration, and tissue remodeling. METHODS: Using primary biopsy and resection specimens from patients with high-risk localized, soft tissue sarcoma treated on a neo/adjuvant chemotherapy study, SPARC expression was assessed and compared to patient and tumor characteristics, treatment, and outcomes. Survival functions were estimated using the Kaplan-Meier method and compared using the log-rank test. The Cox model was used for multivariate analysis. RESULTS: Fifty patients had primary tumor specimens available. High, low, and no SPARC expression was found in 22, 13, and 15 patients, respectively. There was no significant difference in time to recurrence or OS between patients in these three groups. Comparing lack of SPARC expression with any SPARC expression, there was no significant difference in time to recurrence in patients without SPARC expression (n = 15) compared to patients with SPARC expression (n = 35). Likewise, there was no statistically significant difference in OS in patients without SPARC expression versus patients whose tumors expressed SPARC. CONCLUSIONS: Although we did not find a statistically significant difference in time to recurrence and OS in patients with high-risk soft tissue sarcoma, we did identify a trend toward improved time to recurrence and OS in patients whose tumors lacked SPARC expression. However, SPARC did not demonstrate the ability to discern which high-risk patients may have a worse prognosis or greater benefit from chemotherapy. TRIAL REGISTRATION: The trial was registered on September 13, 2005 with ClinicalTrials.gov, number https://clinicaltrials.gov/ct2/show/NCT00189137?term=sarcoma&id=NCT00189137&state1=NA%3AUS%3AMI&phase=1&rank=1 .

Riboflavin and ultraviolet illumination affects selected platelet mRNA transcript amounts differently.

BACKGROUND: Pathogen inactivation (PI) techniques use ultraviolet (UV) illumination with or without a photosensitizer to destroy pathogen RNA and DNA. Although lacking a nucleus and innate DNA transcription, platelets (PLTs) contain RNA and can synthesize proteins. The impact of PI on PLT protein synthesis and function is unknown; altered synthesis may affect overall PLT quality. In this study we determine to what extent PLT RNA is affected by PI. STUDY DESIGN AND METHODS: In a pool-and-split design, paired apheresis PLT concentrates were treated with riboflavin and UV illumination or were left untreated. PLT total RNA and mRNA amounts specific for glycoproteins (GP)IIIa, GPIIb, and GPIb; α-granule proteins PLT factor (PF)4; osteonectin and thrombospondin (TSP); and housekeeping protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined using absorbance and quantitative polymerase chain reaction. RESULTS: After treatment, amounts of all analyzed mRNAs were significantly reduced (p < 0.05), but to different degrees. For GAPDH and PF4, transcripts appeared less susceptible to the treatment, with 70% remaining 1 hour after UV illumination. For GPIIIa and TSP, less than 15% remained after treatment. There was a correlation (R(2) = 0.85) between transcript length and amount of mRNA remaining 1 hour after treatment. Total RNA demonstrated a life span equal to the PLT life span of 10 to 11 days. CONCLUSION: This is the first report of the impact of riboflavin and UV illumination on PLT mRNA. Results suggest that all mRNA present in PLTs is affected by the treatment although the degree of the effect varies among transcripts.

Prognostic impact of tumoral and/or peri-tumoral stromal SPARC expressions after surgery in patients with biliary tract cancer.

BACKGROUND AND OBJECTIVE: SPARC (secreted protein acidic and rich in cysteine) is a matricellular glycoprotein that modulates interactions between tumoral cells and the peri-tumoralstroma. SPARC induces proliferation and invasion in vitro, and is a poor prognostic factor in several gastrointestinal cancers. Herein, we evaluated the prognostic value of tumoral and stromal SPARC expression in patients with biliary tract cancer (BTC) after surgery. METHODS: We examined immunohistochemical patterns of SPARC expression in 110 resected BTC specimens and evaluated the prognostic value using prospectively collected data. RESULTS: SPARC was expressed in tumoral cells in 46 samples (42%) and inperi-tumoralstromain 65 samples (59%). Tumoral SPARC expression was not related to major patient characteristics. Stromal SPARC expression was related to lymph node metastasis, stage, margin status, and tumor location. Overall survival at 5 years after surgery was 34.2%. Stromal SPARC (P < 0.001) and tumoral SPARC (P = 0.048) were associated with poor prognosis. Multivariate analysis revealed invasion into lymphatic system, residual tumor, and stromal SPARC as independent prognostic factors. The hazard ratio for patients with positive stromal SPARC was 3.20 (P < 0.001). CONCLUSION: SPARC expression inperi-tumoralstroma predicts a poor prognosis for patients with BTC after surgery.

Preclinical evaluation of nanoparticle albumin-bound paclitaxel for treatment of pediatric bone sarcoma.

The combination of docetaxel and gemcitabine is frequently used to treat recurrent bone sarcoma. Nanoparticle albumin-bound paclitaxel (nab-paclitaxel) is less toxic and more active than docetaxel or paclitaxel for breast cancer patients. The combination of nab-paclitaxel and gemcitabine has preclinical synergy and is approved to treat pancreatic cancer. We observed growth inhibition and improved survival with nab-paclitaxel in a Ewing sarcoma xenograft, and activity was additive with gemcitabine in an osteosarcoma model. Primary Ewing sarcoma tumors expressed the transport protein SPARC, previously associated with nab-paclitaxel activity. These findings provide rationale for further evaluation of nab-paclitaxel with gemcitabine for bone sarcoma.

Analysis of human alveolar osteoblast behavior on a nano-hydroxyapatite substrate: an in vitro study.

BACKGROUND: Nano-hydroxyapatite (nHA) is a potential ideal biomaterial for bone regeneration. However, studies have yet to characterize the behavior of human osteoblasts derived from alveolar bone on nHA. Thus, the aim of the present study was to evaluate the influence of nHA on the adhesion, proliferation and differentiation of these alveolar bone-derived cells. METHODS: Primary human alveolar osteoblasts were collected from the alveolar ridge of a male periodontal patient during osseous resective surgery and grown on culture plates coated with either polylysine or polylysine with nano-hydroxyapatite (POL/nHA) composite. The cells were grown and observed for 14 days, and then assessed for potential modifications to osteoblasts homeostasis as evaluated by quantitative reverse transcriptase-polymerase chain reaction (real time RT-PCR), scanning electron microscopy and atomic force microscopy. RESULTS: Real time PCR revealed a significant increase in the expression of the selected markers of osteoblast differentiation (bone morphogenetic protein (BMP)-2,-5,-7, ALP, COLL-1A2, OC, ON) in cells grown on the POL/nHA substrate. In addition, as compared with the POL surface, cells grown on the POL/nHA substrate demonstrated better osteoconductive properties, as demonstrated by the increase in adhesion and spreading, likely as a result of the increased surface roughness of the composite. CONCLUSIONS: The increased expression of BMPs and osteoinductive biomarkers suggest that nano-hydroxyapatite may stimulate the proliferation and differentiation of local alveolar osteoblasts and thus encourage bone regeneration at sites of alveolar bone regeneration.

Clinical significance of SPARC gene expression in patients with gastric cancer.

PURPOSE: Secreted protein acidic and rich in cysteine (SPARC) is one of the first known matricellular proteins that modulates interactions between cells and extracellular matrix. Recent studies investigated the clinical significance of SPARC gene expression in the development, progression, and metastasis of cancer. The present study examined the relations of the relative expression of the SPARC gene to clinicopathological factors and overall survival in patients with gastric cancer. METHODS: We studied surgical specimens of cancer tissue and adjacent normal mucosa obtained from 227 patients with previously untreated gastric cancer. The relative expression levels of SPARC mRNA in cancer tissue and in adjacent normal mucosa were measured by quantitative real-time, reverse-transcription polymerase chain reaction. RESULTS: The relative expression level of the SPARC gene was higher in cancer tissue than in adjacent normal mucosa. High expression levels of the SPARC gene were related to serosal invasion (P = 0.046). Overall survival at 5 years differed significantly between patients with high SPARC gene expression and those with low expression (P = 0.006). CONCLUSIONS: Overexpression of the SPARC gene may be a useful independent predictor of outcomes in patients with gastric cancer.

A novel three-dimensional bone chip organ culture.

OBJECTIVES: The objective of this study was to develop a 3D bone chip organ culture model. We aimed to collect in vitro evidence of the ability of vital bone chips to promote new bone formation. MATERIALS AND METHODS: We developed a 3D in vitro hypoxic bone chip organ culture model. Histology of the bone chips was performed before and after culture and immunohistochemistry after 3-week culture. The 3D culture supernatants were tested for the presence of pro-angiogenic growth factors, TGFß1, GADPH, bone alkaline phosphatase, osteocalcin, osteonectin, osteopontin, bone sialoprotein and collagen type I. RESULTS: Histology after culture revealed bone chips in a matrix of fibrin remnants and a fibrous-appearing matter. Collagen type I- and IV-positive structures were also identified. Cells could be seen on the surface of the bone chips, with spindle-shaped cells bridging the bone chip particles. Pro-angiogenic growth factors and TGFß1were detected in the 3D cell culture supernatants. The transcripts for osteocalcin, bone sialoprotein and collagen type I were revealed only via PCR. CONCLUSIONS: Our results indicate that bone chips in our 3D organ culture remain vital and may stimulate the growth of a bone-forming matrix. CLINICAL RELEVANCE: The use of autogenous bone chips for oral and maxillofacial bone augmentation procedures is widespread in clinical practice. The rationale for this is that if bone chips remain vital in vivo, they could provide an environment promoting new bone formation through growth factors and cells. This 3D culture method is an essential tool for investigating the behaviour of bone chips.

The expression, regulation and function of secreted protein, acidic, cysteine-rich in the follicle-luteal transition.

The role of the tissue remodelling protein, secreted protein, acidic, cysteine-rich (SPARC), in key processes (e.g. cell reorganisation and angiogenesis) that occur during the follicle-luteal transition is unknown. Hence, we investigated the regulation of SPARC in luteinsing follicular cells and potential roles of SPARC peptide 2.3 in a physiologically relevant luteal angiogenesis culture system. SPARC protein was detected mainly in the theca layer of bovine pre-ovulatory follicles, but its expression was considerably greater in the corpus haemorrhagicum. Similarly, SPARC protein (western blotting) was up-regulated in luteinising granulosa but not in theca cells during a 6-day culture period. Potential regulatory candidates were investigated in luteinising granulosa cells: LH did not affect SPARC (P>0.05); transforming growth factor (TGF) B1 (P<0.001) dose dependently induced the precocious expression of SPARC and increased final levels: this effect was blocked (P<0.001) by SB505124 (TGFB receptor 1 inhibitor). Additionally, fibronectin, which is deposited during luteal development, increased SPARC (P<0.01). In luteal cells, fibroblast growth factor 2 decreased SPARC (P<0.001) during the first 5 days of culture, while vascular endothelial growth factor A increased its expression (P<0.001). Functionally, KGHK peptide, a SPARC proteolytic fragment, stimulated the formation of endothelial cell networks in a luteal cell culture system (P<0.05) and increased progesterone production (P<0.05). Collectively, these findings indicate that SPARC is intricately regulated by pro-angiogenic and other growth factors together with components of the extracellular matrix during the follicle-luteal transition. Thus, it is possible that SPARC plays an important modulatory role in regulating angiogenesis and progesterone production during luteal development.

Evaluation of graft cell viability-efficacy of piezoelectric versus manual bone scraper technique.

PURPOSE: The aim of the present study was to compare the influence of 2 different bone scrapers with respect to graft quality. MATERIALS AND METHODS: The study was conducted as a prospective, controlled experimental study of patients selected from the outpatient unit of the Department of Oral Surgery and Radiology (Dental Clinic, Medical University, Graz, Austria). Bone samples were obtained during routine lower third molar removal. Both a manual bone scraper (MS) and a piezoelectric device (PD) were used in directly adjacent regions in each case. As variables, the chip morphology, cell viability, and osteogenic differentiation were investigated. For statistical analysis, the Student t test and Fisher's exact test (P < .05) were applied. RESULTS: A total of 20 patients (12 women and 8 men, mean age 28.15 ± 5.8 years) were included in the study. A series of 40 bone samples was obtained during lower third molar removal. MS and PD enabled similar intraoral harvest of bone chips. In vitro outgrowth of adherent cells was found in 90% of the MS and 80% of the PD samples after 7 to 18 days, without statistical significance (P = .67). Similar cell viability of outgrowing cells in both groups was observed (94.7% ± 2.2% in the MS group and 94.1% ± 1.6% in the PD group). Reverse transcriptase-polymerase chain reaction analysis and the staining pattern verified osteopotent cells in both groups. CONCLUSIONS: Both manual and piezoelectric techniques are adequate harvesting technologies for limited intraoral augmentations. Our results did not show an advantage for the piezoelectric device.

Biodentine(TM) induces TGF-ß1 release from human pulp cells and early dental pulp mineralization.

AIM: To assess the ability of a recently developed tricalcium silicate-based cement (Biodentine™) to induce reparative dentine synthesis and to investigate its capacity to modulate pulp cells TGF-ß1 secretion. METHODOLOGY: Biodentine™ was directly applied onto the dental pulp in an entire human tooth culture model. After various culture periods, the interaction of the material with dental pulp tissue was analysed on tissue sections. The effect of increasing surface area of this material on TGF-ß1 secretion was investigated on pulp cell cultures and compared with that of MTA, calcium hydroxide and Xeno(®) III adhesive resin. After performing artificial injuries on pulp cell cultures, the materials eluates were added for 24 h and then TGF-ß1 secretion was quantified by ELISA. Controls were performed by incubating intact cells with the culture medium, while injured cells TGF-ß1 level was used as the baseline value. RESULTS: Biodentine™ induced mineralized foci formation early after its application. The mineralization appeared under the form of osteodentine and expressed markers of odontoblasts. Biodentine™ significantly increased TGF-ß1 secretion from pulp cells (P < 0.03) independently of the contact surface increase. This increase was also observed with calcium hydroxide and MTA, but not with the resinous Xeno(®) III. The statistical analysis showed statistically significant differences between capping materials and the resinous Xeno(®) III (P < 0.001). CONCLUSIONS: When Biodentine™ was applied directly onto the pulp, it induced an early form of reparative dentine synthesis, probably due to a modulation of pulp cell TGF-ß1 secretion.

Beta-tricalcium phosphate/type I collagen cones with or without a barrier membrane in human extraction socket healing: clinical, histologic, histomorphometric, and immunohistochemical evaluation.

The aim of this study was to investigate the healing of human extraction sockets filled with ß-tricalcium phosphate and type I collagen (ß-TCP/Clg) cones with or without a barrier membrane. Twenty patients were divided in two groups: (A) ß-TCP/Clg non-membrane and (B) ß-TCP/Clg + barrier membrane. Clinical examination and biopsies from the grafted sites were collected 9 months later. Bone samples were analyzed using histomorphometry and immunohistochemistry. The horizontal dimension of the alveolar ridge was significantly reduced 9 months after socket preservation in the non-membrane group. There was bone formation with no significant differences between the two groups in the areas occupied by new bone (A = 42.4%; B = 45.3%), marrow (A = 42.7%; B = 35.7%), or residual graft (A = 9.7%; B = 12.5%). Immunohistochemistry revealed osteonectin expression in both groups. Both groups demonstrated sufficient amounts of vital bone and socket morphology to support dental implant placement after the 9-month healing period. A future trial to evaluate the alveolar outcomes at an earlier 6-month time point rather than the 9 months used in this study would be of interest.

The multifaceted role of Matricellular Proteins in health and cancer, as biomarkers and therapeutic targets.

The extracellular matrix (ECM) is composed of a mesh of proteins, proteoglycans, growth factors, and other secretory components. It constitutes the tumor microenvironment along with the endothelial cells, cancer-associated fibroblasts, adipocytes, and immune cells. The proteins of ECM can be functionally classified as adhesive proteins and matricellular proteins (MCP). In the tumor milieu, the ECM plays a major role in tumorigenesis and therapeutic resistance. The current review encompasses thrombospondins, osteonectin, osteopontin, tenascin C, periostin, the CCN family, laminin, biglycan, decorin, mimecan, and galectins. The matrix metalloproteinases (MMPs) are also discussed as they are an integral part of the ECM with versatile functions in the tumor stroma. In this review, the role of these proteins in tumor initiation, growth, invasion and metastasis have been highlighted, with emphasis on their contribution to tumor therapeutic resistance. Further, their potential as biomarkers and therapeutic targets based on existing evidence are discussed. Owing to the recent advancements in protein targeting, the possibility of agents to modulate MCPs in cancer as therapeutic options are discussed.

Novel ceramic bone replacement material Osbone® in a comparative in vitro study with osteoblasts.

OBJECTIVE: Hydroxyapatite (HA) is a very common ceramic material for bone replacement due to its similarity in composition to the mineral phase of natural bone. A recently developed bone graft material is Osbone(®), a synthetic HA ceramic available as porous granules with different sizes and block forms. The goal of this study was to characterise Osbone(®) in vitro in comparison to the already established calcium phosphate-based bone grafts Cerasorb M(®) and Bio-Oss(®). MATERIALS AND METHODS: Adhesion and proliferation of SaOS-2 osteoblasts were evaluated quantitatively by determining DNA content and lactate dehydrogenase (LDH) activity and qualitatively by scanning electron microscopy (SEM). In addition, MTT cell vitality staining was performed to confirm the attachment of viable cells to the different materials. Osteogenic differentiation of the cells was evaluated by means of alkaline phosphatase (ALP) activity quantification as well as by gene expression analysis of osteogenic markers using reverse transcriptase PCR. RESULTS: MTT staining after 1 day of adhesion showed viable cells on all examined materials. DNA content and LDH activity revealed proliferation of osteoblasts on Osbone(®) and Cerasorb M(®), but not on Bio-Oss(®) during cultivation over 28 days. SEM showed a well-spread morphology of cells attached to both Osbone(®) and Cerasorb M(®). We detected an increase of specific ALP activity during cultivation of osteoblasts on Osbone(®) and Cerasorb M(®) as well as expression of the bone-related genes ALP, osteonectin, osteopontin and bone sialoprotein II on both materials. CONCLUSIONS: Osbone(®) granules support proliferation and osteogenic differentiation in vitro and are therefore promising candidates for in vivo applications.

Proteomic Signature of Dysfunctional Circulating Endothelial Colony-Forming Cells of Young Adults.

Background A subpopulation of endothelial progenitor cells called endothelial colony-forming cells (ECFCs) may offer a platform for cellular assessment in clinical studies because of their remarkable angiogenic and expansion potentials in vitro. Despite endothelial cell function being influenced by cardiovascular risk factors, no studies have yet provided a comprehensive proteomic profile to distinguish functional (ie, more angiogenic and expansive cells) versus dysfunctional circulating ECFCs of young adults. The aim of this study was to provide a detailed proteomic comparison between functional and dysfunctional ECFCs. Methods and Results Peripheral blood ECFCs were isolated from 11 subjects (45% men, aged 27±5 years) using Ficoll density gradient centrifugation. ECFCs expressed endothelial and progenitor surface markers and displayed cobblestone-patterned morphology with clonal and angiogenic capacities in vitro. ECFCs were deemed dysfunctional if <1 closed tube formed during the in vitro tube formation assay and proliferation rate was <20%. Hierarchical functional clustering revealed distinct ECFC proteomic signatures between functional and dysfunctional ECFCs with changes in cellular mechanisms involved in exocytosis, vesicle transport, extracellular matrix organization, cell metabolism, and apoptosis. Targeted antiangiogenic proteins in dysfunctional ECFCs included SPARC (secreted protein acidic and rich in cysteine), CD36 (cluster of differentiation 36), LUM (lumican), and PTX3 (pentraxin-related protein PYX3). Conclusions Circulating ECFCs with impaired angiogenesis and expansion capacities have a distinct proteomic profile and significant phenotype changes compared with highly angiogenic endothelial cells. Impaired angiogenesis in dysfunctional ECFCs may underlie the link between endothelial dysfunction and cardiovascular disease risks in young adults.

The role of MMP-9 in the anti-angiogenic effect of secreted protein acidic and rich in cysteine.

BACKGROUND: Secreted protein acidic and rich in cysteine (SPARC), a matricellular glycoprotein, modulates cellular interaction with the extracellular matrix and is capable of altering the growth of various cancers. We therefore sought to determine the effect of SPARC expression on medulloblastoma tumour growth and angiogenesis. METHODS: To this extent, we selected three SPARC full-length cDNA overexpressed clones (Daoy-SP). Consequences of SPARC overexpression were studied in terms of cell growth, angiogenesis using co-culture assay in vitro, dorsal skin-fold chamber assay in vivo, PCR Array for human angiogenic genes, as well as western blotting for angiogenic molecules and tumour growth, in an orthotopic tumour model. RESULTS: The SPARC protein and mRNA levels were increased by approximately three-fold in Daoy-SP cells compared with parental (Daoy-P) and vector (Daoy-EV) controls. Daoy-SP clones reduced tumour cell-induced angiogenesis in vitro and in vivo, and formed small tumours with fewer blood vessels when compared with controls. Matrix metalloprotease-9 (MMP-9) and vascular endothelial growth factor (VEGF) expression were decreased in Daoy-SP clones. Further, inhibition of MMP-9 expression caused SPARC-mediated inhibition of angiogenesis and tumour growth as MMP-9 rescued SPARC-mediated anti-angiogenic effect in vitro and tumour growth inhibition in vivo. CONCLUSION: Overexpression of SPARC decreases angiogenesis, which leads to decreased tumour growth. Further, the role of MMP-9 could be attributed to the anti-angiogenic effect of SPARC.

Expression of osteonectin/secreted protein acidic and rich in cysteine and matrix metalloproteinases in ameloblastoma.

BACKGROUND: Ameloblastoma is the most common clinically-significant epithelial odontogenic tumor, and is considered a benign but locally-aggressive tumor of the craniofacial region. Osteonectin/secreted protein acidic and rich in cysteine (SPARC) is induced in response to a number of biological processes such as tumor growth and metastasis, whereas matrix metalloproteinases (MMPs) degrade the extracellular matrix and participate in various biological processes including tumor invasion and metastasis. We hypothesize that SPARC acts with MMPs for the local invasiveness of ameloblastoma. The aim of this study was to examine the association of SPARC with MMP-1, MMP-2, and MMP-9 in ameloblastoma. METHOD: Immunohistochemical expression of SPARC, MMP-1, MMP-2, and MMP-9 as well as co-expression of SPARC and MMP-9 were examined in a cohort of 23 cases of ameloblastoma. RESULTS: SPARC, MMP-1, -2, and -9 were detected in the cytoplasm of the ameloblastic-like columnar cells and stellate-reticulum-like cells as well as in the stromal tissues of fibroblasts and endothelial cells of our cohort of ameloblastoma patients. Furthermore, co-expression of SPARC and MMP-9 were found in 23 cases of ameloblastoma. This may be the first study to demonstrate that the expression level of SPARC was statistically correlated with MMP-9 but not with MMP-1 or -2 in ameloblastoma. CONCLUSION: Our results suggest a putative association between SPARC and MMPs (especially MMP-9) in ameloblastoma to regulate tumor invasion.

Expression of extracellular matrix proteins in adenomatoid odontogenic tumor.

Altered expression of extracellular matrix (ECM) components has been reported in several pathologies; however, few ECM proteins have been evaluated in adenomatoid odontogenic tumor (AOT). The aim of this study was to analyze the expression and distribution of the ECM proteoglycans: biglycan and decorin; and glycoproteins: osteonectin, osteopontin, bone sialoprotein and osteocalcin in the AOT. Three-micrometer sections from paraffin-embedded specimens were evaluated employing a streptavidin-biotin immunohistochemical method with the antibodies against the proteins previously cited. Only the osteonectin was expressed in the epithelial cells. The eosinophilic amorphous material and the connective tissue showed expression of all components studied. The calcification foci expressed only osteopontin. In conclusion, the low expression of the components studied in neoplastic epithelial cells suggests that the epithelial cells act probably as stimulators of the expression by the stroma, which in turn can act as agonist or antagonist of the tumor growth. These results suggest that the components studied probably have a key role in the biological behavior of the AOT.