Justin B. Maxhimer

Regulation of nitric oxide signalling by thrombospondin 1: implications for anti-angiogenic therapies.

In addition to long-term regulation of angiogenesis, angiogenic growth factor signalling through nitric oxide (NO) acutely controls blood flow and haemostasis. Inhibition of this pathway may account for the hypertensive and pro-thrombotic side effects of the vascular endothelial growth factor antagonists that are currently used for cancer treatment. The first identified endogenous angiogenesis inhibitor, thrombospondin 1, also controls tissue perfusion, haemostasis and radiosensitivity by antagonizing NO signalling. 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 signalling could facilitate development of more selective inhibitors.

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 and CD47 regulate blood pressure and cardiac responses to vasoactive stress.

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.

Thrombospondin-1 and CD47 Limit Cell and Tissue Survival of Radiation Injury

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.

Treatment of liver ischemia-reperfusion injury by limiting thrombospondin-1/CD47 signaling.

BACKGROUND Ischemia-reperfusion (I/R) injury remains a primary complication of transplant surgery, accounting for about 80% of liver transplant failures, and is 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. METHODS Wild-type, TSP1-null, and CD47-null mice underwent liver I/R injury. Wild-type animals were pretreated with CD47 or control antibodies before liver I/R injury. Tissue perfusion via laser Doppler imaging, serum enzymes, histology, and immunohistology were assessed. RESULTS TSP1-null and CD47-null mice subjected to subtotal liver I/R injury showed improved perfusion relative to wild-type mice. Null mice subjected to liver I/R had decreased liver enzyme release and less histologic evidence of injury. Elevated TSP1 expression in liver tissue after I/R injury suggested that preventing its interaction with CD47 could be protective. Thus, pretreatment of wild-type mice using a blocking CD47 antibody improved recovery of tissue perfusion and preserved liver integrity after I/R injury. CONCLUSIONS Tissue survival and perfusion after liver I/R injury are limited by TSP1 and CD47. Targeting CD47 before I/R injury enhances tissue survival and perfusion in a model of liver I/R injury and suggests therapeutics for enhancing organ survival in transplantation surgery.

Central Pancreatectomy for the Resection of Benign or Low Malignant Potential Neoplasms

Central pancreatectomy is an uncommonly performed procedure that may be particularly useful for the removal of benign and low malignant potential lesions in the neck and body of the pancreas. This procedure may have fewer major complications and better preserve endocrine and exocrine function than the more commonly performed pancreaticoduodenectomy or distal pancreatectomy. We report our recent experience with central pancreatectomy and review the literature on this topic.

Potentiation of the anticancer effect of valproic acid, an antiepileptic agent with histone deacetylase inhibitory activity, by the kinase inhibitor Staurosporine or its clinically relevant analogue UCN-01

Histone deacetylase inhibitors (HDACIs) are novel anticancer agents with potent cytotoxicity against a wide range of malignancies. We have previously demonstrated that either Calphostin C (CC) (a protein kinase C (PKC) inhibitor) or Parthenolide (an NF-κB inhibitor) abrogates HDACI-induced transcriptional activation of NF-κB and p21, which is associated with profound potentiation of HDACI-mediated induction of apoptosis. Valproic acid (VA), a commonly used antiepileptic agent, has recently been shown to be an HDACI. This study was aimed to evaluate the anticancer property of VA in thoracic cancer cells and the development of clinically relevant strategies to enhance VA-mediated induction of apoptosis using kinase inhibitors Staurosporine (STP) or its analogue UCN-01. Treating cultured thoracic cancer cells with VA (0.62–10.0 mM) resulted in significant cell line- and dose-dependent growth inhibition (IC50 values: 4.1–6.0 mM) and cell cycle arrest at G1/S checkpoint with profound accumulation of cells at G0/G1 phase but little induction of apoptosis. Valproic acid, being an HDACI, caused significant dose-dependent accumulation of hyperacetylated histones, following 24 h of treatment. Valproic acid-mediated 5–20-fold upregulation of transcriptional activity of NF-κB was substantially (50–90%) suppressed by cotreatment with CC, STP or UCN-01. Whereas minimal death (<20%) was observed in cells treated with either VA (1.0 or 5.0 mM) alone or kinase inhibitors alone, 60–90% of cells underwent apoptosis following exposure to combinations of VA+kinase inhibitors. Kinase inhibitor-mediated suppression of NF-κB transcriptional activity played an important role in sensitising cancer cells to VA as direct inhibition of NF-κB by Parthenolide drastically synergised with VA to induce apoptosis (VA+Parthenolide: 60–90% compared to <20% following single-drug treatments). In conclusion, VA, a well-known antiepileptic drug, has mild growth-inhibitory activity on cultured cancer cells. The weak VA-mediated induction of apoptosis of thoracic cancer cells can be profoundly enhanced either by Parthenolide, a pharmacologic inhibitor of NF-κB, or by UCN-01 a kinase inhibitor that has already undergone phase I clinical development. Combinations of VA with either a PKC inhibitor or an NF-κB inhibitor are promising novel molecularly targeted therapeutics for thoracic cancers.

Thrombospondin-1/CD47 blockade following ischemia-reperfusion injury is tissue protective.

Background: Nitric oxide has prosurvival effects that can limit ischemia-reperfusion injuries. However, the matrix glycoprotein thrombospondin-1 is induced following ischemia-reperfusion injury and limits nitric oxide signaling by engaging its cell surface receptor CD47. In this article, the authors examine whether postinjury blocking of this inhibitory signal can protect from ischemia-reperfusion injury in a rat flap model. Methods: A total of 40 tissue flaps were created in rats based solely on the deep inferior epigastric vessels. Microvascular clamps were used to create 45 minutes of ischemia time to the flaps. The flaps were then treated using a monoclonal antibody to CD47 or an isotype-matched control immunoglobulin G1 5 or 30 minutes after clamp removal. Twenty-four or 72 hours postoperatively, the necrotic area of the flap was determined, and serum, deep inferior epigastric vessels, and flaps were harvested for analysis from five rats in each respective group. Results: Treatment with a CD47 antibody 5 minutes after reperfusion significantly reduces flap necrosis compared with immunoglobulin G1 control (9 percent versus 43 percent; p < 0.01). The protective effect is even more dramatic when treatment is delayed until 30 minutes after reperfusion (10 percent versus 88 percent for control; p < 0.01). Markers of neutrophil and endothelial cell activation along with total leukocytes are reduced in CD47 antibody–treated flaps, as are tissue malondialdehyde levels. Levels of cyclic guanosine monophosphate are elevated 72 hours postoperatively in the CD47 antibody–treated deep inferior epigastric vessels versus the control flaps. Conclusions: Therapies targeting the thrombospondin-1 receptor CD47 offer potential for increasing tissue survival in ischemia-reperfusion injuries. The ability to protect when given after ischemia-reperfusion injury enables a broader clinical applicability.

Rapid and profound potentiation of Apo2L/TRAIL-mediated cytotoxicity and apoptosis in thoracic cancer cells by the histone deacetylase inhibitor Trichostatin A: the essential role of the mitochondria-mediated caspase activation cascade

Apo2L/TRAIL is actively investigated as a novel targeted agent to directly induce apoptosis of susceptible cancer cells. Apo2L/TRAIL-refractory cells can be sensitized to the cytotoxic effect of this ligand by cytotoxic chemotherapeutics. The aim of this study was to evaluate the in vitro tumoricidal activity of the Apo2L/TRAIL + Trichostatin A in cultured thoracic cancer cells and to elucidate the molecular basis of the synergistic cytotoxicity of this combination. Concurrent exposure of cultured cancer cells to sublethal concentrations of Apo2L/TRAIL and Trichostatin A resulted in profound enhancement of Apo2L/TRAIL-mediated cytotoxicity in all cell lines regardless of their intrinsic susceptibility to this ligand. This combination was not toxic to primary normal cells. While Apo2L/TRAIL alone or Trichostatin A alone mediated < 20% cell death, 60 to 90% of cancer cells were apoptotic following treatment with TSA + Apo2L/TRAIL combinations. Complete translocation of Bax from the cytosol to the mitochondria compartment was mainly observed in combination-treated cells and this was correlated with robust elevation of caspase 9 proteolytic activity indicative of activation of the mitochondria apoptogenic effect. Profound TSA + Apo2L/TRAIL–mediated cytotoxicity and apoptosis were completely abrogated by either Bcl2 over-expression or by the selective caspase 9 inhibitor, highlighting the essential role of mitochondria-dependent apoptosis signaling cascade in this process. Moreover, increased caspase 8 activity observed in cells treated with the TSA + Apo2L/TRAIL combination was completely suppressed by Bcl-2 over-expression or by the selective caspase 9 inhibitor indicating that the elevated caspase 8 activity in combination-treated cells was secondary to a mitochondria-mediated amplification feedback loop of caspase activation. These finding form the basis for further development of HDAC inhibitors + Apo2L/TRAIL combination as novel targeted therapy for thoracic malignancies.

Thrombospondin-1 and CD47 signaling regulate healing of thermal injury in mice.

More than 2.5 million Americans suffer from burn injuries annually, and burn management is a major public health problem. Treatments have been developed to manage wound injuries employing skin grafts, various dressings and topical and systemic agents. However, these often achieve limited degrees of success. We previously reported that targeting the interaction of thrombospondin-1 with its signaling receptor CD47 or deletion of the genes encoding either of these proteins in mice improves recovery from soft tissue ischemic injuries as well as tissue injuries caused by ionizing radiation. We now demonstrate that the absence of CD47 improves the rate of wound closure for a focal dermal second-degree thermal injury, whereas lack of thrombospondin-1 initially delays wound closure compared to healing in wild type mice. Doppler analysis of the wounded area showed increased blood flow in both CD47 and thrombospondin-1 null mice. Accelerated wound closure in the CD47 null mice was associated with increased fibrosis as demonstrated by a 4-fold increase in collagen fraction. Wound tissue of CD47 null mice showed increased thrombospondin-1 mRNA and protein expression and TGF-β1 mRNA levels. Activation of latent TGF-β1 was increased in thermally injured CD47-null tissue as assessed by phosphor-ylation of the TGF-β1 receptor-regulated transcription factors SMAD-2 and -3. Overall these results indicate that targeting CD47 may improve the speed of healing thermal injuries, but some level of CD47 expression may be required to limit the long term TGF-β1-dependent fibrosis of these wounds.