Jeff S. Isenberg

The chemical biology of nitric oxide: implications in cellular signaling.

Nitric oxide (NO) has earned the reputation of being a signaling mediator with many diverse and often opposing biological activities. The diversity in response to this simple diatomic molecule comes from the enormous variety of chemical reactions and biological properties associated with it. In the past few years, the importance of steady-state NO concentrations has emerged as a key determinant of its biological function. Precise cellular responses are differentially regulated by specific NO concentration. We propose five basic distinct concentration levels of NO activity: cGMP-mediated processes ([NO]<1-30 nM), Akt phosphorylation ([NO] = 30-100 nM), stabilization of HIF-1alpha ([NO] = 100-300 nM), phosphorylation of p53 ([NO]>400 nM), and nitrosative stress (1 microM). In general, lower NO concentrations promote cell survival and proliferation, whereas higher levels favor cell cycle arrest, apoptosis, and senescence. Free radical interactions will also influence NO signaling. One of the consequences of reactive oxygen species generation is to reduce NO concentrations. This antagonizes the signaling of nitric oxide and in some cases results in converting a cell-cycle arrest profile to a cell survival profile. The resulting reactive nitrogen species that are generated from these reactions can also have biological effects and increase oxidative and nitrosative stress responses. A number of factors determine the formation of NO and its concentration, such as diffusion, consumption, and substrate availability, which are referred to as kinetic determinants for molecular target interactions. These are the chemical and biochemical parameters that shape cellular responses to NO. Herein we discuss signal transduction and the chemical biology of NO in terms of the direct and indirect reactions.

Molecular mechanisms for discrete nitric oxide levels in cancer

Nitric oxide (NO) has been invoked in nearly every normal and pathological condition associated with human physiology. In tumor biology, nitrogen oxides have both positive and negative affects as they have been implicated in both promoting and preventing cancer. Our work has focused on NO chemistry and how it correlates with cytotoxicity and cancer. Toward this end, we have studied both concentration- and time-dependent NO regulation of specific signaling pathways in response to defined nitrosative stress levels that may occur within the tumor microenvironment. Threshold levels of NO required for activation and stabilization of key proteins involved in carcinogenesis including p53, ERK, Akt and HIF have been identified. Importantly, threshold NO levels are further influenced by reactive oxygen species (ROS) including superoxide, which can shift or attenuate NO-mediated signaling as observed in both tumor and endothelial cells. Our studies have been extended to determine levels of NO that are critical during angiogenic response through regulation of the anti-angiogenic agent thrombospondin-1 (TSP-1) and pro-angiogenic agent matrix metalloproteinase-9 (MMP-9). The quantification of redox events at the cellular level has revealed potential mechanisms that may either limit or potentiate tumor growth, and helped define the positive and negative function of nitric oxide in cancer.

Increasing Survival of Ischemic Tissue by Targeting CD47

Thrombospondin-1 (TSP1) limits the angiogenic and vasodilator activities of NO. This activity of TSP1 can be beneficial in some disease states, but endogenous TSP1 limits recovery of tissue perfusion following fixed ischemic injury in dorsal skin flaps in mice. Using mice lacking the TSP1 receptors CD36 or CD47, we now show that CD47 is the necessary receptor for limiting NO-mediated vascular smooth muscle relaxation and tissue survival following ischemic injury in skin flaps and hindlimbs. We further show that blocking CD47 or TSP1 using monoclonal antibodies and decreasing CD47 expression using an antisense morpholino oligonucleotide are effective therapeutic approaches to dramatically increase survival of soft tissue subjected to fixed ischemia. These treatments facilitate rapid vascular remodeling to restore tissue perfusion and increase skin and muscle viability. Thus, limiting CD47-dependent antagonism of NO-mediated vasodilation and vascular remodeling is a promising therapeutic modality to preserve tissues subject to ischemic stress.

Thrombospondin-1 Inhibits Nitric Oxide Signaling via CD36 by Inhibiting Myristic Acid Uptake

Although CD36 is generally recognized to be an inhibitory signaling receptor for thrombospondin-1 (TSP1), the molecular mechanism for transduction of this signal remains unclear. Based on evidence that myristic acid and TSP1 each modulate endothelial cell nitric oxide signaling in a CD36-dependent manner, we examined the ability of TSP1 to modulate the fatty acid translocase activity of CD36. TSP1 and a CD36 antibody that mimics the activity of TSP1 inhibited myristate uptake. Recombinant TSP1 type 1 repeats were weakly inhibitory, but an anti-angiogenic peptide derived from this domain potently inhibited myristate uptake. This peptide also inhibited membrane translocation of the myristoylated CD36 signaling target Fyn and activation of Src family kinases. Myristate uptake stimulated cGMP synthesis via endothelial nitric-oxide synthase and soluble guanylyl cyclase. CD36 ligands blocked myristate-stimulated cGMP accumulation in proportion to their ability to inhibit myristate uptake. TSP1 also inhibited myristate-stimulated cGMP synthesis by engaging its receptor CD47. Myristate stimulated endothelial and vascular smooth muscle cell adhesion on type I collagen via the NO/cGMP pathway, and CD36 ligands that inhibit myristate uptake blocked this response. Therefore, the fatty acid translocase activity of CD36 elicits proangiogenic signaling in vascular cells, and TSP1 inhibits this response by simultaneously inhibiting fatty acid uptake via CD36 and downstream cGMP signaling via CD47.

Differential Interactions of Thrombospondin-1, -2, and -4 with CD47 and Effects on cGMP Signaling and Ischemic Injury Responses

Thrombospondin-1 regulates nitric oxide (NO) signaling in vascular cells via CD47. Because CD47 binding motifs are conserved in the C-terminal signature domains of all five thrombospondins and indirect evidence has implied CD47 interactions with other family members, we compared activities of recombinant signature domains of thrombospondin-1, -2, and -4 to interact with CD47 and modulate cGMP signaling. Signature domains of thrombospondin-2 and -4 were less active than that of thrombospondin-1 for inhibiting binding of radiolabeled signature domain of thrombospondin-1 or SIRPα (signal-regulatory protein) to cells expressing CD47. Consistent with this binding selectivity, the signature domain of thrombospondin-1 was more potent than those of thrombospondin-2 or -4 for inhibiting NO-stimulated cGMP synthesis in vascular smooth muscle cells and downstream effects on cell adhesion. In contrast to thrombospondin-1- and CD47-null cells, primary vascular cells from thrombospondin-2-null mice lack enhanced basal and NO-stimulated cGMP signaling. Effects of endogenous thrombospondin-2 on NO/cGMP signaling could be detected only in thrombospondin-1-null cells. Furthermore, tissue survival of ischemic injury and acute recovery of blood flow in thrombospondin-2-nulls resembles that of wild type mice. Therefore, thrombospondin-1 is the dominant regulator of NO/cGMP signaling via CD47, and its limiting role in acute ischemic injury responses is not shared by thrombospondin-2.

Radioprotection in Normal Tissue and Delayed Tumor Growth by Blockade of CD47 Signaling

Inhibition of a membrane receptor protects normal tissue from radiation injury and simultaneously enhances the ability of ionizing radiation to delay tumor growth. One major caveat of radiation therapy in cancer treatment is that the effective dose delivered to the individual is oftentimes necessarily limited to avoid major side effects that arise from collateral damage inflicted on surrounding normal tissue. Efforts to devise methods to sensitize tumor tissue to radiation injury or to protect normal tissue by scavenging for reactive by-products of radiation have only been moderately successful, as their broad clinical utility is hampered by a lack of specificity or by toxicity. Now, a team of researchers describes an approach to protecting normal human cells from high-dose radiation damage while, at the same time, increasing the sensitivity of tumor cells to radiation. We know that the pathway triggered by the secretory glycoprotein thrombospondin-1 (TSP1) and its corresponding membrane receptor CD47 in response to injury limits the survival of vascular cells and tissues. In this work, Roberts et al. have devised a strategy by which the systemic blockade of TSP1 and its receptor CD47 inhibits downstream signaling functions to protect against radiation injury the highly radiation-sensitive endothelial cells that line the lumen of the vasculature. More important, they show in mice injected with human tumors that suppression of CD47 by systemic administration of an antisense CD47 morpholino can sensitize the tumors to high-dose radiation therapy while keeping collateral damage at bay, exemplified in part by the resilience of the skin, muscle, and bone to radiation injury. Their experiments simultaneously show a significant delay in the time that these tumors take to grow back. This phenomenon remains to be explained, but there is an improvement in vascular function in irradiated, CD47-suppressed mice, and, at the cellular level, in vivo stem cells are still viable and proliferate, whereas peripheral immune cells, which infiltrate the inflammatory tumor microenvironment, are protected and recruited to the site. Although it remains speculative how CD47 participates in antitumor immunity within this experimental context, combined treatment of high-dose radiation and CD47 suppression in these translational studies suggests that a more aggressive therapeutic irradiation strategy with concurrent protection of neighboring normal tissue is possible. Testing the effectiveness of such a treatment strategy will be required to see whether this approach is useful. Radiation-induced damage of normal tissues restricts the therapeutic doses of ionizing radiation that can be delivered to tumors and thereby limits the effectiveness of radiotherapy. Thrombospondin-1 signaling through its cell surface receptor CD47 limits recovery from several types of stress, and mice lacking either gene are profoundly resistant to radiation injury. We describe strategies to protect normal tissues from radiation damage with antibodies to CD47 or thrombospondin-1, a CD47-binding peptide, or antisense suppression of CD47. A morpholino oligonucleotide targeting CD47 confers radioresistance to human endothelial cells in vitro and protects soft tissue, bone marrow, and tumor-associated leukocytes in irradiated mice. In contrast, CD47 suppression in mice bearing melanoma or squamous lung tumors before irradiation results in 89% and 71% smaller tumors, respectively. Thus, inhibition of CD47 signaling maintains the viability of normal tissues after irradiation while increasing the radiosensitivity of tumors.

Thrombospondin-1 supports blood pressure by limiting eNOS activation and endothelial-dependent vasorelaxation

AIMSnThrombospondin-1 (TSP1), via its necessary receptor CD47, inhibits nitric oxide (NO)-stimulated soluble guanylate cyclase activation in vascular smooth muscle cells, and TSP1-null mice have increased shear-dependent blood flow compared with wild-type mice. Yet, the endothelial basement membrane should in theory function as a barrier to diffusion of soluble TSP1 into the arterial smooth muscle cell layer. These findings suggested that endothelial-dependent differences in blood flow in TSP1-null mice may be the result of direct modulation of endothelial NO synthase (eNOS) activation by circulating TSP1. Here we tested the hypothesis that TSP1 inhibits eNOS activation and endothelial-dependent arterial relaxation.nnnMETHODS AND RESULTSnAcetylcholine (ACh)-stimulated activation of eNOS and agonist-driven calcium transients in endothelial cells were inhibited by TSP1. TSP1 also inhibited eNOS phosphorylation at serine(1177). TSP1 treatment of the endothelium of wild-type and TSP1-null but not CD47-null arteries inhibited ACh-stimulated relaxation. TSP1-null vessels demonstrated greater endothelial-dependent vasorelaxation compared with the wild type. Conversely, TSP1-null arteries demonstrated less vasoconstriction to phenylephrine compared with the wild type, which was corrected upon inhibition of eNOS. In TSP1-null mice, intravenous TSP1 blocked ACh-stimulated decreases in blood pressure, and both intravenous TSP1 and a CD47 agonist antibody acutely elevated blood pressure in mice.nnnCONCLUSIONnTSP1, via CD47, inhibits eNOS activation and endothelial-dependent arterial relaxation and limits ACh-driven decreases in blood pressure. Conversely, intravenous TSP1 and a CD47 antibody increase blood pressure. These findings suggest that circulating TSP1, by limiting endogenous NO production, functions as a pressor agent supporting blood pressure.

Blocking Thrombospondin-1/CD47 Signaling Alleviates Deleterious Effects of Aging on Tissue Responses to Ischemia

Objective—Decreased blood flow secondary to peripheral vascular disease underlies a significant number of chronic diseases that account for the majority of morbidity and mortality among the elderly. Blood vessel diameter and blood flow are limited by the matricellular protein thrombospondin-1 (TSP1) through its ability to block responses to the endogenous vasodilator nitric oxide (NO). In this study we investigate the role TSP1 plays in regulating blood flow in the presence of advanced age and atherosclerotic vascular disease. Methods and Results—Mice lacking TSP1 or CD47 show minimal loss of their resistance to ischemic injury with age and increased preservation of tissue perfusion immediately after injury. Treatment of WT and apolipoprotein E–null mice using therapeutic agents that decrease CD47 or enhance NO levels reverses the deleterious effects of age- and diet-induced vasculopathy and results in significantly increased tissue survival in models of ischemia. Conclusion—With increasing age and diet-induced atherosclerotic vascular disease, TSP1 and its receptor CD47 become more limiting for blood flow and tissue survival after ischemic injury. Drugs that limit TSP1/CD47 regulation of blood flow could improve outcomes from surgical interventions in the elderly and ameliorate vascular complications attendant to aging.

Thrombospondin-1: a physiological regulator of nitric oxide signaling

Abstract.Thrombospondin-1 is a secreted protein that modulates vascular cell behavior via several cell surface receptors. In vitro, nanomolar concentrations of thrombospondin-1 are required to alter endothelial and vascular smooth muscle cell adhesion, proliferation, motility, and survival. Yet, much lower levels of thrombospondin-1 are clearly functional in vivo. This discrepancy was explained with the discovery that the potency of thrombospondin-1 increases more than 100-fold in the presence of physiological levels of nitric oxide (NO). Thrombospondin-1 binding to CD47 inhibits NO signaling by preventing cGMP synthesis and activation of its target cGMP-dependent protein kinase. This potent antagonism of NO signaling allows thrombospondin-1 to acutely constrict blood vessels, accelerate platelet aggregation, and if sustained, inhibit angiogenic responses. Acute antagonism of NO signaling by thrombospondin-1 is important for hemostasis but becomes detrimental for tissue survival of ischemic injuries. New therapeutic approaches targeting thrombospondin-1 or CD47 can improve recovery from ischemic injuries and overcome a deficit in NO-responsiveness in aging. (Part of a Multi-author Review)Thrombospondin-1 is secreted protein that modulates vascular cell behavior via several cell surface receptors. In vitro, nanomolar concentrations of thrombospondin-1 are required to alter endothelial and vascular smooth muscle cell adhesion, proliferation, motility, and survival. Yet, much lower levels of thrombospondin-1 are clearly functional in vivo. This discrepancy was explained with the discovery that the potency of thrombospondin-1 increases more than 100-fold in the presence of physiological levels of NO. Thrombospondin-1 binding to CD47 inhibits NO signaling by preventing cGMP synthesis and activation of its target cGMP-dependent protein kinase. This potent antagonism of NO signaling allows thrombospondin-1 to acutely constrict blood vessels, accelerate platelet aggregation and, if sustained, inhibit angiogenic responses. Acute antagonism of NO signaling by thrombospondin-1 is important for hemostasis but becomes detrimental for tissue survival of ischemic injuries. New therapeutic approaches targeting thrombospondin-1 or CD47 can improve recovery from ischemic injuries and overcome a deficit in NO-responsiveness in aging.

Blockade of Thrombospondin-1-CD47 Interactions Prevents Necrosis of Full Thickness Skin Grafts

Background:Skin graft survival and healing requires rapid restoration of blood flow to the avascular graft. Failure or delay in the process of graft vascularization is a significant source of morbidity and mortality. One of the primary regulators of blood flow and vessel growth is nitric oxide (NO). The secreted protein thrombospondin-1 (TSP1) limits NO-stimulated blood flow and growth and composite tissue survival to ischemia. We herein demonstrate a role for TSP1 in regulating full thickness skin graft (FTSG) survival. Methods and Results:FTSG consistently fail in wild type C57BL/6 mice but survive in mice lacking TSP1 or its receptor CD47. Ablation of the TSP1 receptor CD36, however, did not improve FTSG survival. Remarkably, wild type FTSG survived on TSP1 null or CD47 null mice, indicating that TSP1 expression in the wound bed is the primary determinant of graft survival. FTSG survival in wild type mice could be moderately improved by increasing NO flux, but graft survival was increased significantly through antibody blocking of TSP1 binding to CD47 or antisense morpholino oligonucleotide suppression of CD47. Conclusions:TSP1 through CD47 limits skin graft survival. Blocking TSP1 binding or suppressing CD47 expression drastically increases graft survival. The therapeutic applications of this approach could include burn patients and the broader group of people requiring grafts or tissue flaps for closure and reconstruction of complex wounds of diverse etiologies.