We have previously demonstrated that apigenin promotes the expression of antiangiogenic protein thrombospondin-1 (TSP1) via a mechanism driven by mRNA-binding protein HuR. Here, we generated a novel mouse model with whole-body THBS-1 gene knockout on SKH-1 genetic background, which allows studies of UVB-induced acute skin damage and carcinogenesis and tests TSP1 involvement in apigenin's anticancer effects. Apigenin significantly inhibited UVB-induced carcinogenesis in the wild-type (WT) animals but not in TSP1 KO (TKO) mice, suggesting that TSP1 is a critical component of apigenin's chemopreventive function in UVB-induced skin cancer. Importantly, TKO mice presented with the elevated cutaneous inflammation at baseline, which was manifested by increased inflammatory infiltrates (neutrophils and macrophages) and elevated levels of the two key inflammatory cytokines, IL-6 and IL-12. In agreement, maintaining normal TSP1 expression in the UVB-irradiated skin of WT mice using topical apigenin application caused a marked decrease of circulating inflammatory cytokines. Finally, TKO mice showed an altered population dynamics of the bone marrow myeloid progenitor cells (CD11b+), with dramatic expansion of the population of neutrophil progenitors (Ly6ClowLy6Ghigh) compared to the WT control. Our results indicate that the cutaneous tumor suppressor TSP1 is a critical mediator of the in vivo anticancer effect of apigenin in skin, specifically of its anti-inflammatory action.

Endothelial dysfunction, impaired angiogenesis and cellular senescence in type 2 diabetes constitute dominant risk factors for chronic non-healing wounds and other cardiovascular disorders. Studying these phenomena in the context of diabetes and the TSP1-CD-47 signaling dictated the use of the in vitro wound endothelial cultured system and an in vivo PVA sponge model of angiogenesis. Herein we report that diabetes impaired the in vivo sponge angiogenic capacity by decreasing cell proliferation, fibrovascular invasion and capillary density. In contrast, a heightened state of oxidative stress and elevated expression of TSP1 and CD47 both at the mRNA and protein levels were evident in this diabetic sponge model of wound healing. An in vitro culturing system involving wound endothelial cells confirmed the increase in ROS generation and the up-regulation of TSP1-CD47 signaling as a function of diabetes. We also provided evidence that diabetic wound endothelial cells (W-ECs) exhibited a characteristic feature that is consistent with cellular senescence. Indeed, enhanced SA-β-gal activity, cell cycle arrest, increased cell cycle inhibitors (CKIs) p53, p21 and p16 and decreased cell cycle promoters including Cyclin D1 and CDK4/6 were all demonstrated in these cells. The functional consequence of this cascade of events was illustrated by a marked reduction in diabetic endothelial cell proliferation, migration and tube formation. A genetic-based strategy in diabetic W-ECs using CD47 siRNA significantly ameliorated in these cells the excessiveness in oxidative stress, attenuation in angiogenic potential and more importantly the inhibition in cell cycle progression and its companion cellular senescence. To this end, the current data provide evidence linking the overexpression of TSP1-CD47 signaling in diabetes to a number of parameters associated with endothelial dysfunction including impaired angiogenesis, cellular senescence and a heightened state of oxidative stress. Moreover, it may also point to TSP1-CD47 as a potential therapeutic target in the treatment of the aforementioned pathologies.

OBJECTIVE

A fenestrated phenotype is characteristic of liver sinusoidal endothelial cells (LSECs), but liver sinusoids become defenestrated during fibrosis and other liver diseases. Thrombospondin-1 (TSP1) is a matrix glycoprotein with pro-fibrotic effects, and the CD47-binding fragment of TSP1 also has anti-angiogenic effects in endothelial cells. We hypothesized that the CD47-binding fragment of TSP1 could induce defenestration in LSECs through the Rho-Rho kinase (ROCK)-myosin pathway.

METHODS

Freshly isolated rat LSECs were treated with TSP1 or CD47-binding peptides of TSP1. LSEC fenestration was assessed with scanning electron microscopy, and myosin phosphorylation was assessed with immuno-fluorescence.

RESULTS

Treating LSECs with TSP1 caused a dose-dependent loss of fenestrae, and this effect could not be blocked by SB-431542, the TGF-β1 receptor inhibitor. A CD47-binding fragment of TSP1, p4N1, was able to induce defenestration, and a CD47-blocking antibody, B6H12, was able to suppress p4N1-induced defenestration. The p4N1 fragment also caused contraction of fenestra size, correlated with an increase in myosin activation. Pretreatment with Y-237642 (a ROCK inhibitor) prevented p4N1-induced myosin activation and fenestrae decrease. Simvastatin has also been shown to antagonize Rho-ROCK signalling, and we found that simvastatin pretreatment protected LSECs from p4N1-induced myosin activation and defenestration.

CONCLUSIONS

We conclude that CD47 signals through the Rho-ROCK-myosin pathway to induce defenestration in LSECs. In addition, our results show that simvastatin and Y-237642 have a beneficial impact on fenestration in vitro, providing an additional explanation for the efficacy of these compounds for regression of liver fibrosis.

The yeast two-hybrid system was used to reveal the interactions between proteins residing within the cutaneous basement membrane zone and other gene products expressed in cultured human keratinocytes. The proteins of interest included type VII collagen, the predominant component of anchoring fibrils, and laminin 5, a component of anchoring filaments. Although the two-hybrid system was not able to verify a direct interaction between the type VII collagen NC1 domain and the short arm of Lam(beta)3, the type VII collagen NC1 domain (tVII/NC1) and the laminin 5 beta3 chain globular domain VI (lam5/beta3) cDNAs, when used as baits, detected four overlapping cDNA clones encoding thrombospondin 1 (TSP1). The overlapping region of these cDNAs encodes amino acids 400-459, a segment included within a 70 kDa chymotryptic fragment known to bind type V collagen, laminin-1 and other matrix components. The type VII collagen NC1/TSP1 interaction was confirmed by exchanging the vectors, and the interacting domain was mapped by testing a set of both 5' and 3' deletion constructs. The central region of TSP1, when used as a bait in two-hybrid system, showed strong binding to the fibronectin (FN) type III-like repeats 4-7 of type VII collagen NC1 domain. The TSP1 bait also interacted with laminin 5 beta3 chain domain V/III, and the TSP1/laminin 5 beta3 chain interaction was verified by a GST-fusion protein interaction assay. The transcripts encoding TSP1, TSP2, Lam(beta)3 and type VII collagen were abundant in cultured foreskin keratinocytes, and the expression of TSP1 and TSP2 in a wide variety of adult and fetal tissues was confirmed by PCR analysis of multiple tissue cDNA panels. Furthermore, TSP1 type I repeats showed self interaction, and recognized a clone for extracellular matrix protein fibrillin-2. In addition, clones encoding angiogenesis related protein Jagged1 and a platelet enzyme phospholipase scramblase were identified. Thus, the results indicate several previously undetected interactions of TSP1, which is known to be highly expressed during embryonic development, tissue remodeling and wound healing.

Complement activation, an integral arm of innate immunity, may be the critical link to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Whereas we have previously reported elevated anaphylatoxins-complement component 3a (C3a) and complement component 5a (C5a)-in IPF, which interact with TGF-β and augment epithelial injury in vitro, their role in IPF pathogenesis remains unclear. The objective of the current study is to determine the mechanistic role of the binding of C3a/C5a to their respective receptors (C3aR and C5aR) in the progression of lung fibrosis. In normal primary human fetal lung fibroblasts, C3a and C5a induces mesenchymal activation, matrix synthesis, and the expression of their respective receptors. We investigated the role of C3aR and C5aR in lung fibrosis by using bleomycin-injured mice with fibrotic lungs, elevated local C3a and C5a, and overexpression of their receptors via pharmacologic and RNA interference interventions. Histopathologic examination revealed an arrest in disease progression and attenuated lung collagen deposition (Masson's trichrome, hydroxyproline, collagen type I α 1 chain, and collagen type I α 2 chain). Pharmacologic or RNA interference-specific interventions suppressed complement activation (C3a and C5a) and soluble terminal complement complex formation (C5b-9) locally and active TGF-β1 systemically. C3aR/C5aR antagonists suppressed local mRNA expressions of tgfb2, tgfbr1/2, ltbp1/2, serpine1, tsp1, bmp1/4, pdgfbb, igf1, but restored the proteoglycan, dcn Clinically, compared with pathologically normal human subjects, patients with IPF presented local induction of C5aR, local and systemic induction of soluble C5b-9, and amplified expression of C3aR/C5aR in lesions. The blockade of C3aR and C5aR arrested the progression of fibrosis by attenuating local complement activation and TGF-β/bone morphologic protein signaling as well as restoring decorin, which suggests a promising therapeutic strategy for patients with IPF.-Gu, H., Fisher, A. J., Mickler, E. A., Duerson, F., III, Cummings, O. W., Peters-Golden, M., Twigg, H. L., III, Woodruff, T. M., Wilkes, D. S., Vittal, R. Contribution of the anaphylatoxin receptors, C3aR and C5aR, to the pathogenesis of pulmonary fibrosis.

4-Dihydromethyltrisporate dehydrogenase (TDH) converts the (+) mating type sex pheromone 4-dihydromethyltrisporate into methyltrisporate. In Mucor mucedo, this conversion is required only in the (-) mating type. Expression of the TDH encoding TSP1 gene was analyzed qualitatively using reverse-transcribed PCR. TSP1 is constitutively transcribed in the (+) and in the (-) mating type, irrespective of the mating situation. By immunodetection, the translation product is also formed constitutively. In contrast to gene expression, TDH enzyme activity depends on the sexual status of the mycelium. Activity is restricted to the sexually stimulated (-) mating type. Non-stimulated (-), as well as stimulated and non-stimulated (+) mycelia exhibit no activity and do not influence activity in stimulated (-) mycelia. Time course analysis shows strongly increased enzyme activity at 80 min after stimulation. Low activity exists from the onset of stimulation, indicating that additional regulation mechanisms are involved in TDH function.

ADAMTS13 is a 190 kDa zinc protease encoded by a gene located on chromosome 9q34. This protease specifically hydrolyzes von Willebrand factor (VWF) multimers, thus causing VWF size reduction. ADAMTS13 belongs to the A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS) family, involved in proteolytic processing of many matrix proteins. ADAMTS13 consists of numerous domains including a metalloprotease domain, a disintegrin domain, several thrombospondin type 1 (TSP1) repeats, a cysteine-rich domain, a spacer domain and 2 CUB (Complement c1r/c1s, sea Urchin epidermal growth factor, and Bone morphogenetic protein) domains. ADAMTS13 cleaves a single peptide bond (Tyr1605-Met1606) in the central A2 domain of the VWF molecule. This proteolytic cleavage is essential to reduce the size of ultra-large VWF polymers, which, when exposed to high shear stress in the microcirculation, are prone to form with platelets clumps, which cause severe syndromes called thrombotic microangiopathies (TMAs). In this review, we a) discuss the current knowledge of structure-function aspects of ADAMTS13 and its involvement in the pathogenesis of TMAs, b) address the recent findings concerning proteolytic processing of VWF multimers by different proteases, such as the leukocyte-derived serine and metallo-proteases and c) indicate the direction of future investigations.

Diabetes is an important health issue because of its increasing prevalence and association with impaired wound healing. Epidermal keratinocytes with overexpressed antiangiogenic molecule thrombospondin-1 (TSP1) have been shown to impair proper wound healing. This study examined the potential involvement of keratinocyte-derived TSP1 on diabetic wound healing. Cultured human keratinocytes and diabetic rat model were used to evaluate the effect of high-glucose environment on TSP1 expression in epidermal keratinocytes, and the molecular mechanisms involved in the process were also studied. We demonstrated that high-glucose environment increased TSP1 expression in keratinocytes. In addition, increased oxidative stress induced DNA hypomethylation at the TSP1 promoter region in keratinocytes exposed to high-glucose environment. Similar findings were found in our diabetic rat model. Early antioxidant administration normalized TSP1 expression and global DNA methylation status in diabetic rat skin and improved wound healing in vivo. Because oxidative stress contributed to TSP1 DNA hypomethylation, early recognition of diabetic condition and timely administration of antioxidant are logical approaches to reduce complications associated with diabetes as alterations in epigenome may not be reversible by controlling glucose levels during the later stages of disease course.

Thrombospondin-1 (TSP1) is an extracellular matrix glycoproteins that affecting cell adhesion, motility and growth. Based on its effects on tumors, TSP1 is thought to be a potential regulator of tumor growth and metastasis. In this study, we clarified TSP1 immunoreactivity in human esophageal squamous cell carcinoma and its clinicopathological significance. TSP1 immunoreactivity was detected mainly in the cancer stroma and was observed infrequently in cancer cells. According to the TNM classification, 70.6% (12/17) of the T3 esophageal cancers were TSP1-positive, while only 26.9% (7/26) of the Tis and T1 cancers showed TSP1 expression. Lymph node metastasis and venous involvement was frequently found in the TSP1-positive cases (71.4% and 80.0%, respectively) of esophageal squamous cell carcinoma (p < 0.001). This observation suggested that TSP1 expression plays an important role in cancer cell growth and metastasis of human esophageal squamous cell carcinomas, and that stromal TSP1 immunoreactivity is a good predictor of venous involvement and lymph node metastasis.

The presence of unusually large multimers of von Willebrand factor (VWF) is thought to be a major pathogenic factor for thrombotic thrombocytopenic purpura (TTP). ADAMTS13 is a protease that regulates the multimeric size and function of VWF by cleaving VWF. Hence, congenital or acquired deficiency of ADAMTS13 causes life-threatening illness of TTP. Mutations in the ADAMTS13 gene cause inherited TTP, and the development of autoantibodies that inhibit ADAMTS13 activity frequently are associated with acquired TTP. ADAMTS13 consists of 1,427 amino acid residues and is composed of multiple structural and functional domains, containing a signal peptide, a propeptide, a reprolysin-like metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 (Tsp1) motif, a cysteine-rich domain, a spacer domain, seven additional Tsp1 repeats, and two CUB domains. In particular, the cysteine-rich/spacer domains are essential for VWF cleavage and are the principal epitopes recognized by autoantibodies in patients with acquired TTP. Therefore, it is likely that these domains are involved in the recognition and binding of ADAMTS13 to VWF. ADAMTS13 circulates in the blood in an active state, and efficiently cleaves unfold form of VWF induced under shear stress caused by blood flow, preventing the accumulation of pathogenic unusually large VWF multimers (ULVWF). Thus, ADAMTS13 helps maintain vascular homeostasis by preventing the excess thrombus formation.

BACKGROUND

Angiotensin II (Ang II) and transforming growth factor β (TGFβ) are closely involved in the pathogenesis of diabetic complications. We aimed to determine whether an aberrant thrombospondin 1 (TSP1)-mediated TGFβ1/Smads signaling pathway specifically affects vascular fibrosis in diabetic rats and whether valsartan, an Ang II subtype 1 receptor blocker, has an anti-fibrotic effect.

METHODS

Age-matched male Wistar rats were randomly divided into 3 groups: control (n = 8), diabetes (n = 16) and valsartan (30 mg/kg/day) (n = 16). Type 2 diabetes mellitus (T2DM) was induced by a high-calorie diet and streptozotocin injection. Morphological and biomechanical properties of the thoracic aorta were assessed by echocardiography and cardiac catheterization. Masson staining was used for histological evaluation of extracellular matrix (ECM). The expression of components in the TSP1-mediated TGFβ1/Smads signaling pathway was analyzed by immunohistochemistry and real-time quantitative reverse transcription polymerase chain reaction.

RESULTS

As compared with controls, diabetic aortas showed reduced distensibility and compliance, with excess ECM deposition. Components in the TSP1-mediated TGFβ1/Smads signaling pathway, including TSP1, TGFβ1, TGFβ type II receptor (TβRII), Smad2 and Smad3, were accumulated in vascular smooth muscle cytoplasm of diabetic aortas and their protein and mRNA levels were upregulated. All these abnormalities were attenuated by valsartan.

CONCLUSIONS

TSP1-mediated TGFβ1/Smads pathway activation plays an important role in marcovascular remodeling in T2DM in rat. Valsartan can block the pathway and ameliorate vascular fibrosis.

UNASSIGNED

The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1053842818141195.

The thrombospondins (TSPs) are a family of extracellular glycoproteins that display distinct patterns of temporal and spatial expression during development. In this study, we investigated the expression of two of the TSPs--TPS1 and TSP2--during the course of differentiation of embryonal carcinoma cells in vitro. We report that both TSP1 and TSP2 mRNA and protein synthesis are induced during the differentiation of P19EC cells into neurons, glial cells, and fibroblasts. Immunofluorescence studies indicate that TSP1 displays a fibrillar pattern of staining, characteristic of an extracellular matrix protein, in differentiated P19EC cells. In contrast, TSP2 is cell-associated and is present on differentiated P19EC cells and on primary neurons and glial cells obtained from a 17-day embryonic mouse cerebral cortex. Interestingly, although both TSP1 and TSP2 are more prevalent in areas of differentiated cells, they display distinct patterns of deposition. These observations suggest that TSP1 and TSP2 may function differently during neurogenesis. The response of TSP1 and TSP2 to differentiation of P19EC cells indicates that this cell system will serve as a valuable model for the study of TSP expression and function during neurogenesis.

Oxaliplatin-based chemotherapy is the main treatment regimen for gastric cancer (GC), but can fail because of drug resistance. We investigated the role of a recently identified drug-resistance gene, taxol-resistant gene 1 (Txr1), in oxaliplatin resistance. A retrospective study based on banked tissue was carried out. We collected clinical data from 95 patients with stage II-III GC who were treated with radical D2 surgery and standardized first-line chemotherapy with oxaliplatin; paraffin blocks of their tumor specimens were prepared for a tissue microarray in which Txr1 expression was analyzed immunohistochemically and compared with their clinical data and their 3-year disease-free survival (DFS) rate. The human GC cell line, SGC7901, was developed into the oxaliplatin-resistant cell line, SGC7901/L-OHP, using slowly increased oxaliplatin concentrations over 6 months. The relationship between Txr1 expression and drug-resistance of oxaliplatin in GC was studied with drug intervention, gene silencing technology, real-time PCR and Western blot analysis. Of the 95 patients with GC, those with TXR1(-) GC had longer postoperative 3-year DFS (77.8 %) than those with TXR1(+) GC (52.9 %). In oxaliplatin-resistant SGC7901/L-OHP cells, the main expression location of Txr1 shifted from the nucleus to cytoplasm, and both the mRNA and protein expression of Txr1 were higher than that of the parental cells, whereas expression of thrombospondin-1 (TSP1) decreased. When the Txr1 gene was silenced, TSP1 expression increased and the oxaliplatin resistance was significantly reduced in SGC7901/L-OHP cells. Changed Txr1 expression in GC affects the efficacy of oxaliplatin-based chemotherapy. Increased Txr1 expression decreases TSP1 expression and inhibits apoptosis. Txr1 could be a target in reversing oxaliplatin resistance in GC.

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant neoplasms worldwide. Patients are often diagnosed at advanced stages with poor prognosis due to the absence of obvious early symptoms. Here, we applied a high-throughput serum peptidome analysis to identify circulating peptide markers of ESCC. Weak cationic exchange magnetic beads coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used for two-stage proteotypic peptide profiling in complex serum samples collected from 477 cancer patients and healthy controls. We established a genetic algorithm model containing three significantly differentially expressed peptides at 1,925.5, 2,950.6 and 5,900.0 Da with a sensitivity and specificity of 97.00% and 95.92% in the training set and 97.03% and 100.00% in the validation set, respectively. The model's diagnostic capability was significantly better than SCC-Ag and Cyfra 21-1, especially for early stage ESCC, with an achieved sensitivity of 96.94%. Subsequently, these peptides were identified as fragments of AHSG, TSP1 and FGA by linear ion trap-orbitrap hybrid tandem mass spectrometry. Notably, increased tissue and serum levels of TSP1 in ESCC were verified and correlated with disease progression. In addition, tissue TSP1 was an independent poor prognostic factor in ESCC. In conclusion, the newly established circulating peptide panel and identified proteins could serve as potential biomarkers for the early detection and diagnosis of ESCC. Nevertheless, a larger cohort will be required for further unequivocal validation of their clinical application.

The purpose of this study was to determine the role of taxol resistance gene 1 (TXR1) in a taxol-resistant breast cancer cell line. Expression levels of TXR1 and thrombospondin-1 (TSP1) were detected by RT-PCR and Western blot analysis. In MCF-7 cells transfected with TXR1 plasmids, the expression of a number of drug resistance genes was monitored, as well as cell proliferation. MCF-7 cells were also transiently transfected with a chemically synthesized small interfering RNA (siRNA) targeting TXR1. Taxol concentrations varying from 0.6 to 6 μg/ml were shown to inhibit the proliferation of MCF-7 cells in a time- and dose-dependent manner by arresting cells in the G2/M phase. Additionally, 0.06 μg/ml taxol was used to establish a taxol-resistant MCF-7 cell line, MCF-7/T. When TXR1 was exogenously expressed, a taxol-resistant phenotype was further induced and the expression levels of BCRP, GSTP1 and MVP increased. Transient transfection of TXR1-targeting siRNAs was shown to restore the chemosensitivity of MCF-7 cells. These results suggest that an increased expression of TXR1 is a novel mechanism for reversing the taxol resistance of breast cancer cells.

Accumulating evidence suggests that thrombospondin 1 (TSP1) is an important player in diabetic nephropathy. However, the role of TSP1 in podocyte injury and the development of non-diabetic proteinuric kidney disease is largely unknown. In the current study, by using a well-established podocyte injury model (adriamycin-induced nephropathy mouse model), we examined the contribution of TSP1 to the development of proteinuric kidney disease. We found that TSP1 was up-regulated in the glomeruli, notably in podocytes, in adriamycin injected mice before the onset of proteinuria. ADR treatment also stimulated TSP1 expression in cultured human podocytes in vitro. Moreover, increased TSP1 mediated ADR-induced podocyte apoptosis and actin cytoskeleton disorganization. This TSP1's effect was through a CD36-dependent mechanism and involved in the stimulation of p38MAPK pathway. Importantly, in vivo data demonstrated that TSP1 deficiency protected mice from ADR induced podocyte loss and foot process effacement. ADR induced proteinuria, glomerulosclerosis, renal macrophage infiltration and inflammation was also attenuated in TSP1 deficient mice. Taken together, these studies provide new evidence that TSP1 contributes to the development of non-diabetic proteinuric kidney disease by stimulating podocyte injury and the progression of renal inflammation.

Fibrosis of normal tissues often accompanies radiation treatment of cancer. Activation of the transforming growth factor-beta (TGF-beta) signaling pathway is thought to play a major role in radiation-induced fibrosis and has prompted the development and assessment of low molecular weight inhibitors of the pathway. Previous studies with halofuginone have shown it to inhibit TGF-beta signaling in vitro and protect mice from radiation-induced leg contraction (a model for soft tissue fibrosis). The current study confirms these findings for HaCaT cells stimulated with exogenous TGF-beta treatment. Reducing the halifuginone treatment from 7 days/week (used previously) to 5 days/week post-radiation exposure provided significant protection against radiation-induced leg contraction in mice 3 and 4 months post-radiation treatment. Halofuginone treatment was shown to attenuate TGF-beta signaling molecules taken from irradiated skin including TGF-betaRII, pSmad3, Smad7, and TSP1. The latter, TSP1, a co-activator of TGF-beta may serve as a suitable biomarker for monitoring the efficacy of halofuginone should it be evaluated in a clinical setting for protection against radiation-induced fibrosis.

We have previously shown that thrombosponsin-1 (TSP1) and PECAM-1 are components of a regulatory switch whose reciprocal regulation in the endothelial cells (EC) promotes an angiogenic or a differentiated, quiescent phenotype. The physiological role TSP1 plays in modulation of PECAM-1 expression and function during vascular development and angiogenesis remains largely unknown. Here we demonstrate that PECAM-1 undergoes alternative splicing in its cytoplasmic domain generating eight isoforms in the retinal vasculature of wild type and TSP1-/- mice. All PECAM-1 isoforms examined contained exon 13. The frequency of PECAM-1 isoform(s) containing exon 14 was significantly higher during early stages of retinal vascularization, which decreased during later stages of retinal vascularization in wild type mice. In contrast, the frequency of exon 14 containing PECAM-1 isoform(s) did not significantly change during retinal vascularization in TSP1-/- mice. They consistently expressed higher number of isoforms with exon 14 during later stages of retinal vascularization. The higher level of PECAM-1 isoforms with exon 14 was also observed in cultured TSP1-/- retinal EC compared to wild type retinal EC. This was consistent with increased amounts of Src and SHP-2 associated with PECAM-1, and enhanced migration and proliferation in TSP1-/- retinal EC. These data suggest PECAM-1 signaling in the endothelium is modulated by its alternative splicing during retinal vascular development and angiogenesis, which may be impacted by TSP1 expression.

The extracellular matrix (ECM) plays a key role in the modulation of cancer cell invasion. In urothelial carcinoma of the bladder (UC) the role of ECM proteins has been widely studied. The mechanisms, which are involved in the development of invasion, progression and generalization, are complex, depending on the interaction of ECM proteins with each other as well as with cancer cells. The following review will focus on the pathogenetic role and prognostic value of structural proteins, such as laminins, collagens, fibronectin (FN), tenascin (Tn-C) and thrombospondin 1 (TSP1) in UC. In addition, the role of integrins mediating the interaction of ECM molecules and cancer cells will be addressed, since integrin-mediated FN, Tn-C and TSP1 interactions seem to play an important role during tumor cell invasion and angiogenesis.

Thrombospondin-1 (TSP1) is a multifunctional matricellular protein known to promote progression of chronic kidney disease. To gain insight into the underlying mechanisms through which TSP1 accelerates chronic kidney disease, we compared disease progression in Col4a3 knockout (KO) mice, which develop spontaneous kidney failure, with that of Col4a3;Tsp1 double-knockout (DKO) mice. Decline of excretory renal function was significantly delayed in the absence of TSP1. Although Col4a3;Tsp1 DKO mice did progress toward end-stage renal failure, their kidneys exhibited distinct histopathological lesions, compared with creatinine level-matched Col4a3 KO mice. Although kidneys of both Col4a3 KO and Col4a3;Tsp1 DKO mice exhibited a widened tubulointerstitium, predominant lesions in Col4a3 KO kidneys were collagen deposition and fibroblast accumulation, whereas in Col4a3;Tsp1 DKO kidney inflammation was predominant, with less collagen deposition. Altered disease progression correlated with impaired activation of transforming growth factor-β1 (TGF-β1) in vivo and in vitro in the absence of TSP1. In summary, our findings suggest that TSP1 contributes to progression of chronic kidney disease by catalyzing activation of latent TGF-β1, resulting in promotion of a fibroproliferative response over an inflammatory response. Furthermore, the findings suggest that fibroproliferative and inflammatory lesions are independent entities, both of which contribute to decline of renal function.

CD47 is a transmembrane protein that functions as a receptor for thrombospondin-1 (TSP1) and a ligand for inhibitory receptor signal-regulatory protein-α (SIRPα). Blocking the interaction between CD47 on tumor cells and SIRPα on macrophages has been shown to induce antitumor responses. Here we investigated the role of CD47 expression in tumor stroma in tumorigenesis by comparing tumor growth in wild-type (WT) and CD47-deficient mice after subcutaneous injection of syngeneic prostate cancer cells. We found that CD47 deficiency in tumor stromal endothelial cells enhances angiogenesis, leading to suppressed tumor necrosis formation and accelerated tumor progression. Tumors from CD47-deficient mice also showed improved vascular integrity and stability, as well as increased expression of vascular endothelial growth factor (VEGF)-A and VEGF receptor 2 (VEGFR2) compared to those from WT mice. Moreover, reduced macrophage recruitment, likely due to decreased TSP1 production, was detected in tumors from CD47-deficient mice. Our results indicate that although treatment with antibody against CD47 induces antitumor immune responses by blocking the inhibitory CD47-SIRPα signaling, this treatment may also potentially promote tumor progression by blocking CD47 signaling in tumor stromal endothelial cells.

Previously, we demonstrated that upstream stimulatory factor 2 (USF2) mediates high glucose-induced thrombospondin1 (TSP1) gene expression and TGF-beta activity in glomerular mesangial cells and plays a role in diabetic renal complications. In the present studies, we further determined the molecular mechanisms by which high glucose levels regulate USF2 gene expression. In primary rat mesangial cells, we found that glucose treatment time and dose-dependently up-regulated USF2 expression (mRNA and protein). By using cycloheximide to block the de novo protein synthesis, similar rate of USF2 degradation was found under either normal glucose or high glucose conditions. USF2 mRNA stability was not altered by high glucose treatment. Furthermore, high glucose treatment stimulated USF2 gene promoter activity. By using the luciferase-promoter deletion assay, site-directed mutagenesis, and transactivation assay, we identified a glucose-responsive element in the USF2 gene promoter (-1,740 to -1,620, relative to the transcription start site) and demonstrated that glucose-induced USF2 expression is mediated through a cAMP-response element-binding protein (CREB)-dependent transactivation of the USF2 promoter. Furthermore, siRNA-mediated CREB knock down abolished glucose-induced USF2 expression. Taken together, these data indicate that high glucose levels up-regulate USF2 gene transcription in mesangial cells through CREB-dependent transactivation of the USF2 promoter.

Latrophilin adhesion-GPCRs (Lphn1-3 or ADGRL1-3) and Unc5 cell guidance receptors (Unc5A-D) interact with FLRT proteins (FLRT1-3), thereby promoting cell adhesion and repulsion, respectively. How the three proteins interact and function simultaneously is poorly understood. We show that Unc5D interacts with FLRT2 in cis, controlling cell adhesion in response to externally presented Lphn3. The ectodomains of the three proteins bind cooperatively. Crystal structures of the ternary complex formed by the extracellular domains reveal that Lphn3 dimerizes when bound to FLRT2:Unc5, resulting in a stoichiometry of 1:1:2 (FLRT2:Unc5D:Lphn3). This 1:1:2 complex further dimerizes to form a larger 'super-complex' (2:2:4), using a previously undescribed binding motif in the Unc5D TSP1 domain. Molecular dynamics simulations, point-directed mutagenesis and mass spectrometry demonstrate the stability and molecular properties of these complexes. Our data exemplify how receptors increase their functional repertoire by forming different context-dependent higher-order complexes.

OBJECTIVE

To review the current literature regarding the role of matricellular proteins in glaucoma, specifically in the lamina cribrosa (LC) region of the optic nerve head (ONH) and the trabecular meshwork (TM).

METHODS

A literature search was performed for published articles describing the expression and function of matricellular proteins such as thrombospondin (TSP), connective tissue growth factor (CTGF), secreted protein acidic and rich in cysteine (SPARC), and periostin in glaucoma.

RESULTS

In glaucoma, there are characteristic extracellular matrix (ECM) changes associated with optic disc cupping in the ONH and subsequent visual field defects. Matricellular proteins are a family of nonstructural secreted glycoproteins, which enable cells to communicate with their surrounding ECM, including CTGF, also known as CCN2, TSPs, SPARC, periostin, osteonectin, and tenascin-C and -X, and other ECM proteins. Such proteins appear to play a role in fibrosis and increased ECM deposition. Importantly, most are widely expressed in tissues particularly in the TM and ONH, and deficiency of TSP1 and SPARC has been shown to lower intraocular pressure in mouse models of glaucoma through enhanced outflow facility.

CONCLUSIONS

This article highlights the role of matricellular proteins in glaucoma pathology. The potential role of these proteins in glaucoma is emerging as some have an association with the pathophysiology of the TM and LC region and might therefore be potential targets for therapeutic intervention in glaucoma.