In addition to long term regulation of angiogenesis, angiogenic growth factor signaling through nitric oxide (NO) acutely controls blood flow and hemostasis. Inhibition of this pathway may account for the hypertensive and prothrombotic side effects of vascular endothelial growth factor antagonists currently used for cancer treatment. The first identified endogenous angiogenesis inhibitor, thrombospondin-1, also controls tissue perfusion, hemostasis, and radiosensitivity by antagonizing NO signaling. We examine the role of these and other emerging activities of thrombospondin-1 in cancer. Clarifying how endogenous and therapeutic angiogenesis inhibitors regulate vascular NO signaling could facilitate development of more selective inhibitors.The insight that tumor growth requires angiogenesis led to the development and clinical use of angiogenesis inhibitors as cancer therapeutics. The drugs bevacizumab, sorafenib, and sunitinib, which are approved by the US Food and Drug Administration for the treatment of several cancers, act specifically or in part by blocking the angiogenic activity of the vascular endothelial growth factor (VEGF) pathway. These targeted drugs significantly extend survival of cancer patients but have cardiovascular side effects that include hypertension [G] and thrombosis [G] [1][2][3] . Although most attempts to define the etiology of their hypertensive activity have focused on long term changes in vessel architecture, VEGF signaling via nitric oxide (NO) also has acute effects on vessel tone [G] 4,5 , and hypertension induced by the experimental VEGF receptor kinase inhibitor cediranib was recently shown to be caused by acute disruption of NO synthesis in vascular endothelium 6 . Recent studies of the first identified endogenous angiogenesis inhibitor, thrombospondin-1 (TSP1), reveal that it also inhibits NO-mediated signaling to acutely control tissue perfusion [G] and hemostasis [G] 7,8 .Interestingly, the pioneering work of Folkman and colleagues showed that tumors can produce circulating angiogenesis inhibitors 9 , and circulating TSP1 levels are elevated in people and mice with certain cancers [10][11][12] . The benefit to the tumor of circulating angiogenesis inhibitors, which in some cases are produced by stromal rather than tumor cells, is unclear. We propose that elevated plasma TSP1 can enhance tumor perfusion through its hypertensive activity. This review synthesizes emerging evidence that hemostasis and tissue blood flow are acute targets of both endogenous and therapeutic angiogenesis inhibitors and explores ways that this insight can be used to improve anti-angiogenic therapy. Nitric oxidePhysiological activity of NO was first described by Davy in 1800 13 , but its production by mammalian tissues and role as a signaling molecule in vascular cells was not discovered until the 1980s 14 . The primary endogenous source of NO in endothelial cells is the endothelial isoform of nitric oxide synthase [G] (eNOS, also known as NOS3). eNOS is a highly regulated enzyme th...

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 using CD47 or thrombospondin-1 antibodies, 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 tumorassociated leukocytes in irradiated mice. In contrast, CD47 suppression in mice bearing melanoma or squamous lung tumors prior to irradiation result in 89% and 71% smaller tumors, respectively. Thus, inhibiting CD47 signaling maintains the viability of normal tissues following irradiation while increasing the radiosensitivity of tumors.

The molecular mechanisms of heparan sulfate proteoglycan downregulation in the glomerular basement membrane (GBM) of the kidneys with diabetic nephropathy remain controversial. In the present study, we showed that the expression of heparanase-1 (HPR1), a heparan sulfate-degrading endoglycosidase, was upregulated in the renal epithelial cells in the kidney with diabetic nephropathy. Urinary HPR1 levels were elevated in patients with diabetic nephropathy. In vitro cell culture studies revealed that HPR1 promoterdriven luciferase reporter gene expression, HPR1 mRNA, and protein were upregulated in renal epithelial cells under high glucose conditions. Induction of HPR1 expression by high glucose led to decreased cell surface heparan sulfate expression. HPR1 inhibitors were able to restore cell surface heparan sulfate expression. Functional analysis revealed that renal epithelial cells grown under high glucose conditions resulted in an increase of basement membrane permeability to albumin. Our studies suggest that loss of heparan sulfate in the GBM with diabetic nephropathy is attributable to accelerated heparan sulfate degradation by increased HPR1 expression. Diabetes 54:2172-2178, 2005 D iabetic nephropathy is a major cause of endstage renal disease. It is characterized by glomerular hemodynamic abnormalities that result in glomerular hyperfiltration, leading to glomerular damage as evidenced by microalbuminuria. As glomerular function continues to decline, overt proteinuria, decreased glomerular filtration rate, and end-stage renal failure result. The glomerular basement membrane (GBM) functions as a primary barrier to allow molecules to selectively cross over into the urinary space (1). The GBM is a specialized extracellular matrix produced as a thin sheet-like structure by glomerular epithelial cells (1). The main components in the GBM are collagen type IV, laminin, and heparan sulfate proteoglycans (HSPGs) (1). HSPGs are composed of a protein core attached with side-chains of the complex glycosaminoglycan heparan sulfate (2). Because of their negative charge, heparan sulfate chains are highly hydrated and thus play a key space-filling and molecular-sieving role in the GBM. It has been long recognized that ultrastructural changes, including GBM thickening, mesangial expansion, and reduction in HSPGs, lead to the loss of charge selectivity and altered glomerular size selectivity, allowing albumin leakage into the urinary space.Heparanase-1 (HPR1) is an endoglycosidase that specifically degrades HSPGs. Several lines of evidence suggest that HPR1 plays a critical role in the pathogenesis of proteinuria in diabetic nephropathy: 1) heparin and other glycosaminoglycans, which have been recently identified as heparanase inhibitors (3), can slow down diabetic nephropathy in preclinical and clinical settings (4); 2) recent studies demonstrated that glomerular charge selectivity is greatly compromised in mice treated with bacterial heparinase and other glycosaminoglycan-degrading enzymes (5); and 3) overexpression of HPR...

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Background-Ischemia-reperfusion (I/R) injury remains a primary complication of transplant surgery, accounting for ∼80% of liver transplant failures, and a major source of morbidity in other pathologic conditions. Activation of endothelium and inflammatory cell recruitment are central to the initiation and promulgation of I/R injury, which can be limited by the bioactive gas nitric oxide (NO). The discovery that thrombsospondin-1 (TSP1), via CD47, limits NO signaling in vascular cells and ischemic injuries in vivo suggested that I/R injury could be another important target of this signaling pathway.

Nitric oxide (NO) locally regulates vascular resistance and blood pressure by modulating blood vessel tone. Thrombospondin-1 signaling via its receptor CD47 locally limits the ability of NO to relax vascular smooth muscle cells and increase regional blood flow in ischemic tissues. To determine whether thrombospondin-1 plays a broader role in central cardiovascular physiology, we examined vasoactive stress responses in mice lacking thrombospondin-1 or CD47. Mice lacking thrombospondin-1 exhibit activity-associated increases in heart rate, central diastolic and mean arterial blood pressure and a constant decrease in pulse pressure. CD47-deficient mice have normal central pulse pressure but elevated resting peripheral blood pressure. Both null mice show exaggerated decreases in peripheral blood pressure and increased cardiac output and ejection fraction in response to NO. Autonomic blockade also induces exaggerated hypotensive responses in awake thrombospondin-1 null and CD47 null mice. Both null mice exhibit a greater hypotensive response to isoflurane, and autonomic blockage under isoflurane anesthesia leads to premature death of thrombospondin-1 null mice. Conversely, the hypertensive response to epinephrine is attenuated in thrombospondin-1 null mice. Thus, the matricellular protein thrombospondin-1 and its receptor CD47 serve as acute physiological regulators of blood pressure and exert a vasopressor activity to maintain global hemodynamics under stress.

Radiation, a primary mode of cancer therapy, acutely damages cellular macromolecules and DNA and elicits stress responses that lead to cell death. The known cytoprotective activity of nitric oxide (NO) is blocked by thrombospondin-1, a potent antagonist of NO/ cGMP signaling in ischemic soft tissues, suggesting that thrombospondin-1 signaling via its receptor CD47 could correspondingly increase radiosensitivity. We show here that soft tissues in thrombospondin-1-null mice are remarkably resistant to radiation injury. Twelve hours after 25-Gy hindlimb irradiation, thrombospondin-1-null mice showed significantly less cell death in both muscle and bone marrow. Two months after irradiation, skin and muscle units in null mice showed minimal histological evidence of radiation injury and near full retention of mitochondrial function. Additionally , both tissue perfusion and acute vascular responses to NO were preserved in irradiated thrombospondin-1-null hindlimbs. The role of thrombospondin-1 in radiosensitization is specific because thrombospondin-2-null mice were not protected. However , mice lacking CD47 showed radioresistance similar to thrombospondin-1-null mice. Both thrombospondin-1-and CD47-dependent radiosensitization is cell autonomous because vascular cells isolated from the respective null mice showed dramatically increased survival and improved proliferative capacity after irradiation in vitro. Therefore , thrombospondin-1/CD47 antagonists may have selective radioprotective activity for normal tissues.

Background-Nitric oxide has pro-survival effects that can limit ischemia-reperfusion (I/R) injuries. However, the matrix glycoprotein thrombospondin-1 is induced following I/R injury and limits nitric oxide signaling by engaging its cell surface receptor CD47. We herein examine whether post-injury blocking of this inhibitory signal can protect from I/R injury in a rat flap model.