Nitric oxide (NO) drives pro-survival responses in vascular cells and limits platelet adhesion, enhancing blood flow and minimizing thrombosis. The matricellular protein thrombospondin-1 (TSP1), through interaction with its receptor CD47, inhibits soluble guanylyl cyclase (sGC) activation by NO in vascular cells. In vascular smooth muscle cells (VSMCs) both intracellular cGMP and cAMP regulate adhesion, contractility, proliferation, and migration. cGMP can regulate cAMP through feedback control of hydrolysis. Inhibition of the cAMP phosphodiesterase-4 selectively interfered with the ability of exogenous TSP1 to block NO-driven VSMC adhesion but not cGMP accumulation, suggesting that cAMP also contributes to VSMC regulation by TSP1. Inhibition of phosphodiesterase-4 was sufficient to elevate cAMP levels, and inhibiting guanylyl cyclase or phosphodiesterase-3, or adding exogenous TSP1 reversed this increase in cAMP. Thus, TSP1 regulates VSMC cAMP levels in part via cGMP-dependent inhibition of phosphodiesterase-3. Additionally basal cAMP levels were consistently elevated in both VSMCs and skeletal muscle from TSP1 null mice, and treating null cells with exogenous TSP1 suppressed cAMP levels to those of wild type cells. TSP1 inhibited both forskolin and isoproterenol stimulated increases in cAMP in VSMCs. TSP1 also abrogated forskolin and isoproterenol stimulated vasodilation. Consistent with its ability to directly limit adenylyl cyclase-activated vasodilation, TSP1 also limited cAMP-induced dephosphorylation of myosin light chain-2. These findings demonstrate that TSP1 limits both cGMP and cAMP signaling pathways and functional responses in VSMCs and arteries, by both phosphodiesterase-dependent cross talk between these second messengers and by inhibition of adenylyl cyclase activation.

Hyperactivity and disturbances of attention are common behavioral disorders whose underlying cellular and neural circuit causes are not understood. We report the discovery that striatal astrocytes drive such phenotypes through a hitherto unknown synaptic mechanism. We found that striatal medium spiny neurons (MSNs) triggered astrocyte signaling via γ-aminobutyric acid B (GABA_B) receptors. Selective chemogenetic activation of this pathway in striatal astrocytes in vivo resulted in acute behavioral hyperactivity and disrupted attention. Such responses also resulted in upregulation of the synaptogenic cue thrombospondin-1 (TSP1) in astrocytes, increased excitatory synapses, enhanced corticostriatal synaptic transmission, and increased MSN action potential firing in vivo. All of these changes were reversed by blocking TSP1 effects. Our data identify a form of bidirectional neuron-astrocyte communication and demonstrate that acute reactivation of a single latent astrocyte synaptogenic cue alters striatal circuits controlling behavior, revealing astrocytes and the TSP1 pathway as therapeutic targets in hyperactivity, attention deficit, and related psychiatric disorders.

CD47, a receptor for thrombospondin-1, limits two important regulatory axes: nitric oxide-cGMP signaling and cAMP signaling, both of which can promote mitochondrial biogenesis. Electron microscopy revealed increased mitochondrial densities in skeletal muscle from both CD47 null and thrombospondin-1 null mice. We further assessed the mitochondria status of CD47-null vs WT mice. Quantitative RT-PCR of RNA extracted from tissues of 3 month old mice revealed dramatically elevated expression of mRNAs encoding mitochondrial proteins and PGC-1α in both fast and slow-twitch skeletal muscle from CD47-null mice, but modest to no elevation in other tissues. These observations were confirmed by Western blotting of mitochondrial proteins. Relative amounts of electron transport enzymes and ATP/O(2) ratios of isolated mitochondria were not different between mitochondria from CD47-null and WT cells. Young CD47-null mice displayed enhanced treadmill endurance relative to WTs and CD47-null gastrocnemius had undergone fiber type switching to a slow-twitch pattern of myoglobin and myosin heavy chain expression. In 12 month old mice, both skeletal muscle mitochondrial volume density and endurance had decreased to wild type levels. Expression of myosin heavy chain isoforms and myoglobin also reverted to a fast twitch pattern in gastrocnemius. Both CD47 and TSP1 null mice are leaner than WTs, use less oxygen and produce less heat than WT mice. CD47-null cells produce substantially less reactive oxygen species than WT cells. These data indicate that loss of signaling from the TSP1-CD47 system promotes accumulation of normally functioning mitochondria in a tissue-specific and age-dependent fashion leading to enhanced physical performance, lower reactive oxygen species production and more efficient metabolism.

CD47 and thrombospondin 1 and 2 (TSP1 and TSP2) expression were analysed by real-time reverse transcription polymerase chain reaction in fluorescence-activated cell sorted plasma cells (PCs) from patients at consecutive stages of multiple myeloma (MM) development. 80% of MM patients, but only 39% of patients with monoclonal gammopathy of undetermined significance (MGUS) expressed CD47; median expression level increased 10-fold with progression from MGUS to MM. Elevated TSP1/TSP2 levels occurred in bone marrow cultures from MM patients compared with healthy donors. CD47 and TSP1/TSP2 may have a potential role in the pathophysiology of MM, probably in the interaction between MM PCs and the microenvironment.

OBJECTIVE

To isolate and characterize primary retinal astrocytes in culture (RAC) from wild-type and transgenic mice to aid the study of their properties in vitro.

METHODS

Astrocytes were isolated from wild-type and transgenic Immortomice by collagenase digestion of the retina. Affinity purification using magnetic beads coated with anti-platelet endothelial cell adhesion molecule-1 (PECAM-1) was used to remove retinal endothelial cells. The remaining cells were cultured and expanded. The majority of these cells were identified as astrocytes. These cells were characterized for expression of astrocytic markers using fluorescence-activated cell sorting (FACS) and immunostaining analysis. The expression of various integrins and other cell adhesion molecules on the surface of retinal astrocytes, their adhesion to various matrix proteins, their migration, and their ability to organize on Matrigel were determined.

RESULTS

Here we describe a method for the isolation of RAC from wild-type and thrombospondin-1 deficient (TSP1-/-) mice. Our results indicated that nearly 100% of cells isolated expressed the astrocytic markers GFAP, NG2, Pax2, and vimentin. These cells were successfully passaged and maintained in culture for several months without a significant loss in expression of astrocytic markers. The RAC expressed alphavbeta3 integrin and other cell adhesion molecules on their surface. The TSP1-/- RAC adhered more strongly to fibronectin and vitronectin compared to the wild-type cells, while neither cell types adhered to collagen and laminin. Wild-type and TSP1-/- RAC exhibited similar migratory characteristics despite alterations in their adhesive properties and production of various matrix proteins. Also, these cells, like endothelial cells, similarly organized into a network in Matrigel.

CONCLUSIONS

The RAC can be readily obtained from wild-type and transgenic mice. This facilitates the comparison and identification of specific gene functions in RAC compared to astrocytes prepared from other sites of central nervous system.

Thrombospondin-1 (TSP1) is a multi-domain, multi-functional glycoprotein synthesized by many cells. Matricellular TSP1 modulates cell adhesion and proliferation. TSP1 is involved in angiogenesis, inflammation, wound healing and cancer. As a major platelet protein, for a long time it was postulated to control hemostasis via platelet aggregate stabilization. However, these in vitro findings have been questioned in the absence of corroborating clinical data and of obvious hemostatic defects in TSP1 gene-deficient mice.Yet, the past few years have provided indices to implicate TSP1 in hemostasis. In clinical studies, a correlation exists between a welldefined TSP1 polymorphism and a significant risk of myocardial infarction. At the same time, recent in vivo animal model data imply TSP1 in the multimer size control of von Willebrand factor, in smooth muscle cell regulation and in vascular perfusion. These findings shed new light on the role of TSP1 in hemostasis and prothrombotic vascular pathologies. (Part of a Multi-author Review).

OBJECTIVE

Thrombospondin-1 (TSP1) and TSP2 are matricellular proteins that have been shown to regulate cytoskeleton, cell adhesion, and extracellular matrix remodeling. Both TSP1 and TSP2 are found in the trabecular meshwork (TM). In cadaver eyes with primary open-angle glaucoma (POAG), TSP1 is increased in one third of patients. We hypothesized that TSP1 and TSP2 participate in the regulation of intraocular pressure (IOP). Methods. IOPs of TSP1-null, TSP2-null mice, and their corresponding wild-type (WT) mice were measured using a commercial rebound tonometer. Fluorophotometric measurements assessed aqueous turnover. Central corneal thickness (CCT) was measured by optical coherence tomography. Iridocorneal angles were examined using light microscopy (LM), immunofluorescence (IF), and transmission electron microscopy (TEM).

RESULTS

Average IOPs of TSP1-null and TSP2-null mice were 10% and 7% less than that of the corresponding WT mice, respectively. CCTs were 6.5% less in TSP1-null mice (P < 0.05) and 1.1% less in TSP2-null mice (P>> 0.05). Fluorophotometric measurements suggest that aqueous turnover rates in TSP1-null and TSP2-null mice are greater than those of WT mice. LM of the TSP1-null and TSP2-null iridocorneal angles reveals morphology, which is indistinguishable from that of their corresponding WTs. IF revealed possible concurrent underexpression of TSP2 in TSP1-null mice and of TSP1 in TSP2-null mice. TEM revealed larger collagen fibril diameters in TSP1-null and TSP2-null mice compared with WTs.

CONCLUSIONS

TSP1-null and TSP2-null mice have lower IOPs than their WT counterparts. The rate of aqueous turnover suggests that the mechanism is enhanced outflow facility. An alteration in the extracellular matrix may contribute to this finding.

Glaucoma is an optic neuropathy affecting approximately 60million people worldwide and is the second most common cause of irreversible blindness. Elevated intraocular pressure (IOP) is the main risk factor for developing glaucoma and is caused by impaired aqueous humor drainage through the trabecular meshwork (TM) and Schlemm's canal (SC). In primary open angle glaucoma (POAG), this elevation in IOP in turn leads to deformation at the optic nerve head (ONH) specifically at the lamina cribrosa (LC) region where there is also a deposition of extracellular matrix (ECM) molecules such as collagen and fibronectin. Matricellular proteins are non-structural secreted glycoproteins that help cells communicate with their surrounding ECM. This family of proteins includes connective tissue growth factor (CTGF), also known as CCN2, thrombospondins (TSPs), secreted protein acidic and rich in cysteine (SPARC), periostin, osteonectin, and Tenascin-C and -X and other ECM proteins. All members appear to play a role in fibrosis and increased ECM deposition. Most are widely expressed in tissues particularly in the TM and ONH and deficiency of TSP1 and SPARC have been shown to lower IOP in mouse models of glaucoma through enhanced outflow facility. The role of these proteins in glaucoma is emerging as some have an association with the pathophysiology of the TM and LC regions and might therefore be potential targets for therapeutic intervention in glaucoma.

We have reexamined the role of endogenous thrombospondin-1 (TSP1) in growth and motility of vascular smooth muscle cells (SMCs). Based on the ability of aortic-derived SMCs isolated from TSP1 null mice and grown in the absence of exogenous TSP1 to grow at comparable rates and to a slightly higher density than equivalent cells from wild-type mice, TSP1 is not necessary for their growth. Low concentrations of exogenous TSP1 stimulate growth of TSP1 null SMCs, but higher doses of TSP1 or its C-terminal domain are inhibitory. However, SMCs from TSP1 null mice are selectively deficient in chemotactic and proliferative responses to platelet-derived growth factor and in outgrowth in three-dimensional cultures. Recombinant portions of the N- and C-terminal domains of TSP1 stimulate SMC chemotaxis through different integrin receptors. Based on these data, the relative deficiency in SMC outgrowth during an ex vivo angiogenic response of muscle tissue from TSP1 null mice is probably due to restriction of platelet-derived growth factor dependent SMC migration and/or proliferation.

The diverse and occasionally conflicting properties described for the extracellular, cell surface-associated protein thrombospondin (TSP) have raised the possibility that functionally distinct forms of the protein exist in the same organism. We have isolated and characterized a partial cDNA clone for mouse TSP that is clearly homologous to, but distinct from, the coding sequence for mouse TSP deduced from a mouse genomic clone (Bornstein, P., Alfi, D., Devarayalu, L., Framson, P., and Li, P. (1990) J. Biol. Chem. 265, 16691-16698). This second TSP, which we term thrombospondin 2, is the product of a separate gene (Thbs2) and is expressed in a variety of mouse tissues in a pattern that differs from that for TSP1. Based on their translated amino acid sequences, it seems likely that TSP1 and TSP2 will be found to have both common and unique properties and that the functional consequences of TSP production will reflect the ratio of the levels of these two related proteins.

Thrombospondin-1 (TSP1) binding to calreticulin (CRT) on the cell surface stimulates association of CRT with LDL receptor-related protein (LRP1) to signal focal adhesion disassembly and engagement of cellular activities. The structural basis for this phenomenon is unknown. We studied the binding thermodynamics of the TSP1-CRT complex and the conformational changes in CRT induced by binding to TSP1 with combined binding free energy analysis, molecular dynamics simulation, and anisotropic network model restrained molecular dynamics simulation. Results showed that mutations of Lys 24 and Lys 32 in TSP1 to Ala and of amino acids 24-26 and 32-34 in CRT to Ala significantly weakened the binding of TSP1 and CRT, which is consistent with experimental results. Upon validation of the calculated binding affinity changes of the TSP1-CRT complex by mutations in key residues in TSP1 and CRT with the experimental results, we performed conformational analyses to understand the role of TSP1 binding to CRT in the induction of conformational changes in CRT. Conformational analyses showed that TSP1 binding to CRT resulted in a more "open" conformation and a significant rotational change for the CRT N-domain with respect to the CRT P-domain, which could expose the potential binding site(s) in CRT for binding to LRP1 to signal focal adhesion disassembly. Results offer structural insight into the role of TSP1 binding to CRT in CRT-induced focal adhesion disassembly.

The membrane attack complex (MAC)/perforin-like protein complement component 9 (C9) is the major component of the MAC, a multi-protein complex that forms pores in the membrane of target pathogens. In contrast to homologous proteins such as perforin and the cholesterol-dependent cytolysins (CDCs), all of which require the membrane for oligomerisation, C9 assembles directly onto the nascent MAC from solution. However, the molecular mechanism of MAC assembly remains to be understood. Here we present the 8 Å cryo-EM structure of a soluble form of the poly-C9 component of the MAC. These data reveal a 22-fold symmetrical arrangement of C9 molecules that yield an 88-strand pore-forming β-barrel. The N-terminal thrombospondin-1 (TSP1) domain forms an unexpectedly extensive part of the oligomerisation interface, thus likely facilitating solution-based assembly. These TSP1 interactions may also explain how additional C9 subunits can be recruited to the growing MAC subsequent to membrane insertion.

Interactions between RNA binding proteins (RBPs) and genes are not well understood, especially in regulation of angiogenesis. The RBP HuR binds to the AU-rich (ARE) regions of labile mRNAs, facilitating their translation into protein and has been hypothesized to be a tumor-maintenance gene. Elevated levels of cytoplasmic HuR directly correlate with increased invasiveness and poor prognosis for many cancers, including those of the breast. HuR controls the expression of multiple genes involved in angiogenesis including VEGFα, HIF1α and thrombospondin 1 (TSP1). We investigated the role of HuR in estrogen receptor negative (ER(-)) breast cancer. MDA-MB-231 cells with higher levels of HuR have alterations in cell cycle kinetics and faster growth. Unexpectedly, HuR overexpression significantly interfered with tumor growth in orthotopic mouse models. The putative mechanism seems to be an anti-angiogenetic effect by increasing expression of TSP1 but also surprisingly, downregulating VEGF, a target which HuR normally increases. Our findings reveal that HuR may be regulating a cluster of genes involved in blood vessel formation which controls tumor angiogenesis. An approach of modulating HuR levels may overcome limitations associated with monotherapies targeting tumor vessel formation.

Human and non-human primate salivas retard the infectivity of HIV-1 in vitro and in vivo. Because thrombospondin 1 (TSP1), a high molecular weight trimeric glycoprotein, is concentrated in saliva and can inhibit the infectivity of diverse pathogens in vitro, we sought to determine the role of TSP1 in suppression of HIV infectivity. Sequence analysis revealed a TSP1 recognition motif, previously defined for the CD36 gene family of cell adhesion receptors, in conserved regions flanking the disulfide-linked cysteine residues of the V3 loop of HIV envelope glycoprotein gp120, important for HIV binding to its high affinity cellular receptor CD4. Using solid-phase in vitro binding assays, we demonstrate direct binding of radiolabeled TSP1 to immobilized recombinant gp120. Based on peptide blocking experiments, the TSP1-gp120 interaction involves CSVTCG sequences in the type 1 properdin-like repeats of TSP1, the known binding site for CD36. TSP1 and fusion proteins derived from CD36-related TSP1-binding domains were able to compete with radiolabeled soluble CD4 binding to immobilized gp120. In parallel, purified TSP1 inhibited HIV-1 infection of peripheral blood mononuclear cells and transformed T and promonocytic cell lines. Levels of TSP1 required for both viral aggregation and direct blockade of HIV-1 infection were physiologic, and affinity depletion of salivary TSP1 abrogated >70% of the inhibitory effect of whole saliva on HIV infectivity. Characterization of TSP1-gp120 binding specificity suggests a mechanism for direct blockade of HIV infectivity that might be exploited to retard HIV transmission that occurs via mucosal routes.

We have evaluated the basis for the constitutive and serum-regulated expression of the mouse thrombospondin (TSP) gene in both transiently and stably transfected NIH-3T3 cells. Experiments with deleted and mutated mouse promoter/CAT constructs and gel mobility assays demonstrated that an Egr-1 binding site in the proximal promoter, flanked by overlapping GC boxes and an adjacent GC-rich region, functioned to positively regulate the constitutive activity of the gene. These motifs, and their cognate transcription factors, appear to act in concert, with partial redundancy, so that discrete mutations were only partially effective in reducing transcriptional activity. The Egr-1 site corresponds in position to an NF-Y binding site which functions synergistically with a distal serum-response element to mediate the serum response of the human TSP1 gene. However, neither the Egr-1 motif nor the surrounding proximal promoter region upstream from the TATA box participates in the serum response of mouse TSP1. These experiments add support to the growing realization that similar physiologic responses of homologous genes in mouse and man need not utilize similarly placed cis-acting elements.

Thrombospondin 1 and thrombospondin 2 (TSP1 and TSP2), which comprise the subgroup A thrombospondins, are matricellular proteins. As matricellular proteins, they modulate interactions between cells and the cellular environment, regulate cell adhesion and typically are expressed during tissue formative processes. In general, TSP1 and TSP2 counter angiogenesis (including tumour angiogenesis) and play important but contrasting roles during cutaneous repair. The two proteins are involved in development, including that of the eye, although evidence suggests that they have their greatest impact during tissue production in the adult. In the normal adult eye, they tend to be found at sites of ongoing matrix synthesis or cell-matrix interactions. At these sites, the two proteins possibly influence cellular differentiation and/or basement membrane deposition. TSP1 is also present in the intraocular fluids and drainage pathway, where it may function in maintaining the anti-angiogenic environment and in intraocular pressure control, respectively. TSP1 could also be involved in ocular immune privilege. Unlike in skin wounds, where TSP1 is derived from the blood and is present only in the early phases of repair, ocular tissue damage appears to lead to protacted TSP1 synthesis by local cells. This response might help suppress angiogenesis in the transparent tissues of the eye and so lessen visual axis opacification following injury. However, TSP2, which is also produced by damaged ophthalmic tissue and may be especially important in matrix organisation, seems to augment contraction in anomalous intraocular fibrosis. Elucidating the roles of TSP1 and TSP2 in ocular physiology and pathobiology may lead to improved therapies for neovascular, neoplastic, reparative and other ophthalmic diseases.

Thrombospondin 1 (TSP1) is a multifunctional protein able to activate TGFbeta and to inhibit angiogenesis in vivo. Although usually thought of as an inhibitor of tumor growth, TSP1 may sometimes be present at high levels during tumor progression, suggesting that tumors can eventually overcome their anti-tumor effects. Using a tet-repressible expression system, we demonstrate that murine TSP1 delayed the onset of tumor growth when produced in the tumor bed by rat fibrosarcoma tumor cells or by stromal fibroblasts coinjected with unmodified C6 glioma tumor cells. Yet upon prolonged exposure to TSP1, tumors came to grow at the same rate in the presence as in the absence of TSP1 and transplantation experiments showed that they had become insensitive to inhibition by TSP1 in both syngeneic and immune compromised hosts. Tumor resistance to TSP1 developed as a result of the in vivo outgrowth of pre-existing tumor cell variants that (1) secreted increased amounts of angiogenic factors that counterbalanced the inhibitory effect of TSP1 on neovascularization and (2) grew more efficiently in the presence of TSP1-activated TGFbeta. These results indicate that prolonged and continuous local delivery of a single multifunctional angiogenesis inhibitor like TSP1 to fast-growing tumors can lead to tumor resistance in vivo by fostering the outgrowth of subpopulations that are a by-product of the genetic instability of the tumor cells themselves.

Antiangiogenic thrombospondin-1 (TSP1) induces endothelial cell death via a CD95-mediated cascade. We used this signaling pathway, where CD95/Fas is a rate-limiting intermediate, as a target to optimize the efficacy of TSP1 active peptide, DI-TSP. Like TSP1, DI-TSP upregulated endothelial CD95L in vivo. To modulate CD95 levels, we chose chemotherapy agent doxorubicin (DXR). DXR caused sustained upregulation of CD95 in the activated endothelium at 1/100 of the maximal tolerated dose. DI-TSP and DXR synergistically induced endothelial apoptosis in vitro, and in vivo, in developing murine vessels. Fas decoy, TSP1 receptor antibody and Pifithrin, a p53 inhibitor, severely decreased apoptosis and restored angiogenesis by DXR-DI-TSP combination, evidencing critical roles of CD95 and TSP1. Combined therapy synergistically blocked neovascularization and progression of the bladder and prostate carcinoma. Such informed design of a complex antiangiogenic therapy based on the rate-limiting molecular targets is a novel concept, which may yield new approaches to cancer treatment.

Specific immunotherapy (SIT) is the only specific remedy for the treatment of allergic diseases currently. B cells are important immune cells in the immunity. The role of B cells in immune regulatory activities has not been fully understood yet. This study aims to elucidate the role of the thrombospondin (TSP)1-producing B cells in the immune regulatory role of SIT. The results showed that after SIT, the frequency of CD35(+) B cells was increased in the intestine of mice with food allergy. The CD35(+) B cells expressed TSP1 after exposure to specific antigens. Co-culture with the TSP1-producing CD35(+) B cells decreased the levels of CD80/CD86 in dendritic cells; the cells convert naïve CD4(+) T cells to regulatory T cells to inhibit allergic inflammation in the intestine.

ADAMTS-13, a plasma reprolysin-like metalloprotease, cleaves von Willebrand factor (VWF). Severe deficiency of plasma ADAMTS-13 activity results in thrombotic thrombocytopenic purpura (TTP), while mild to moderate deficiencies of plasma ADAMTS-13 activity are emerging risk factors for developing myocardial and cerebral infarction, pre-eclampsia, and malignant malaria. Moreover, Adamts13(-/-) mice develop more severe inflammatory responses, leading to increased ischemia/perfusion injury and formation of atherosclerosis. Structure-function studies demonstrate that the N-terminal portion of ADAMTS-13 (MDTCS) is necessary and sufficient for proteolytic cleavage of VWF under various conditions and attenuation of arterial/venous thrombosis after oxidative injury. The more distal portion of ADAMTS-13 (TSP1 2-8 repeats and CUB domains) may function as a disulfide bond reductase to prevent an elongation of ultra-large VWF strings on activated endothelial cells and inhibit platelet adhesion/aggregation on collagen surface under flow. Remarkably, the proteolytic cleavage of VWF by ADAMTS-13 is accelerated by FVIII and platelets under fluid shear stress. A disruption of the interactions between FVIII (or platelet glycoprotein 1bα) and VWF dramatically impairs ADAMTS-13-dependent proteolysis of VWF in vitro and in vivo. These results suggest that FVIII and platelets may be physiological cofactors regulating VWF proteolysis. Finally, the structure-function and autoantibody mapping studies allow us to identify an ADAMTS-13 variant with increased specific activity but reduced inhibition by autoantibodies in patients with acquired TTP. Together, these findings provide novel insight into the mechanism of VWF proteolysis and tools for the therapy of acquired TTP and perhaps other arterial thrombotic disorders.

Nitric oxide (NO) plays important physiological roles in the vasculature to regulate angiogenesis, blood flow, and hemostasis. In solid tumors, NO is generally acknowledged to mediate angiogenic responses to several growth factors. This contrasts with conflicting evidence that NO can acutely increase tumor perfusion through local vasodilation or diminish perfusion by preferential relaxation of peripheral vascular beds outside the tumor. Because thrombospondin 1 (TSP1) is an important physiological antagonist of NO in vascular cells, we examined whether, in addition to inhibiting tumor angiogenesis, TSP1 can acutely regulate tumor blood flow. We assessed this activity of TSP1 in the context of perfusion responses to NO as a vasodilator and epinephrine as a vasoconstrictor. Nitric oxide treatment of wild type and TSP1 null mice decreased perfusion of a syngeneic melanoma, whereas epinephrine transiently increased tumor perfusion. Acute vasoactive responses were also independent of the level of tumor-expressed TSP1 in a melanoma xenograft, but recovery of basal perfusion was modulated by TSP1 expression. In contrast, overexpression of truncated TSP1 lacking part of its CD47 binding domain lacked this modulating activity. These data indicate that TSP1 primarily regulates long-term vascular responses in tumors, in part, because the tumor vasculature has a limited capacity to acutely respond to vasoactive agents.

Cardiovascular homeostasis and health is maintained through the balanced interactions of cardiac generated blood flow and cross-talk between the cellular components that comprise blood vessels. Central to this cross-talk is endothelial generated nitric oxide (NO) that stimulates relaxation of the contractile vascular smooth muscle (VSMC) layer of blood vessels. In cardiovascular disease this balanced interaction is disrupted and NO signaling is lost. Work over the last several years indicates that regulation of NO is much more complex than previously believed. It is now apparent that the secreted protein thrombospondin-1 (TSP1), that is upregulated in cardiovascular disease and animal models of the same, on activating cell surface receptor CD47, redundantly inhibits NO production and NO signaling. This inhibitory event has implications for baseline and disease-related responses mediated by NO. Further work has identified that TSP1-CD47 signaling stimulates enzymatic reactive oxygen species (ROS) production to further limit blood flow and promote vascular disease. Herein consideration is given to the most recent discoveries in this regard which identify the TSP1-CD47 axis as a major proximate governor of cardiovascular health.

Chronic behavioral stress and beta-adrenergic signaling have been shown to promote cancer progression, whose underlying mechanisms are largely unclear, especially the involvement of epigenetic regulation. Histone deacetylase-2 (HDAC2), an epigenetic regulator, is critical for stress-induced cardiac hypertrophy. It is unknown whether it is necessary for beta-adrenergic signaling-promoted cancer progression. Using xenograft models, we showed that chronic behavioral stress and beta-adrenergic signaling promote angiogenesis and prostate cancer progression. HDAC2 was induced by beta-adrenergic signaling in vitro and in mouse xenografts. We next uncovered that HDAC2 is a direct target of cAMP response element-binding protein (CREB) that is activated by beta-adrenergic signaling. Notably, HDAC2 is necessary for beta-adrenergic signaling to induce angiogenesis. We further demonstrated that, upon CREB activation, HDAC2 represses thrombospondin-1 (TSP1), a potent angiogenesis inhibitor, through epigenetic regulation. Together, these data establish a novel pathway that HDAC2 and TSP1 act downstream of CREB activation in beta-adrenergic signaling to promote cancer progression.

In this study we examined the mechanisms by which glomerular mesangial cells ingest apoptotic cells and the mesangial cell response to this event, since there is in vivo evidence that such semiprofessional phagocytes participate in phagocytic clearance of both apoptotic leukocytes and apoptotic resident cells from inflamed glomeruli, thereby promoting resolution of glomerulonephritis. Mesangial cell phagocytosis of apoptotic neutrophils in vitro was not affected by inhibitors of lectin-like receptors, phosphatidylserine receptors, the 61D3 Ag, and beta1 and beta2 integrins, receptors which have been implicated in phagocytosis of apoptotic cells by particular populations of semiprofessional and professional phagocytes. However, the specific inhibitory effects of cationic aminosugars, Arg-Gly-Asp-Ser (RGDS) peptide, and mAbs to phagocyte alpha(v)beta3 vitronectin receptor integrin and "bridging" thrombospondin 1 (TSP1) indicated that mesangial cell phagocytosis of apoptotic cells involved an alpha(v)beta3/TSP mechanism akin to that described for human monocyte-derived macrophages (Mphi) in which Mphi CD36 plays an important role in binding "bridging" TSP1. However, mesangial cells did not express CD36 and there was no evidence for involvement of alternative phagocyte receptors for TSP1, heparan sulfate proteoglycan and sulfatides. Nevertheless, phagocytosis of apoptotic neutrophils by either mesangial cells or Mphi failed to elicit secretion of IL-8 and MCP-1, representatives of each major class of proinflammatory chemotactic cytokines. We conclude that mesangial cell phagocytosis of apoptotic neutrophils involves a novel CD36-independent, alpha(v)beta3/TSP-mediated mechanism that is uncoupled from chemokine secretion, emphasizing the injury-limiting potential of mesangial cell phagocytosis of apoptotic cells.