Martin J. Romeo

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.

CSF proteome: a protein repository for potential biomarker identification

Proteomic analysis is not limited to the analysis of serum or tissues. Synovial, peritoneal, pericardial and cerebrospinal fluid represent unique proteomes for disease diagnosis and prognosis. In particular, cerebrospinal fluid serves as a rich source of putative biomarkers that are not solely limited to neurologic disorders. Peptides, proteolytic fragments and antibodies are capable of crossing the blood–brain barrier, thus providing a repository of pathologic information. Proteomic technologies such as immunoblotting, isoelectric focusing, 2D gel electrophoresis and mass spectrometry have proven useful for deciphering this unique proteome. Cerebrospinal fluid proteins are generally less abundant than their corresponding serum counterparts, necessitating the development and use of sensitive analytical techniques. This review highlights some of the promising areas of cerebrospinal fluid proteomic research and their clinical applications.

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.

Gene Silencing of CD47 and Antibody Ligation of Thrombospondin-1 Enhance Ischemic Tissue Survival in a Porcine Model: Implications for Human Disease

Background:Insufficient tissue perfusion underlies many acute and chronic diseases. Tissue perfusion in turn requires adequate blood flow, determined in large part by the relative state of relaxation or constriction of arterial vessels. Nitric oxide (NO) produced by vascular cells modulates blood flow and tissue perfusion by relaxing and dilating arteries. Recently, we reported that the secreted protein thrombospondin-1 (TSP1), through its cell surface receptor CD47, limits the ability of NO to relax and dilate blood vessels and thus decreases tissue perfusion. In the present study, we tested the hypothesis that blocking TSP1-CD47 signaling increases ischemic tissue survival in random cutaneous porcine flaps. Methods:Random cutaneous flaps 2 × 10 cm2 were raised in white hairless Yucatan miniature pigs and were treated with a monoclonal antibody to TSP1, an antisense morpholino oligonucleotide to CD47 or control agents and tissue survival assessed. Primary vascular smooth muscle cell cultured from Yucatan pigs were also treated with the same agents ± and an NO donor (DEA/NO) and cGMP quantified. Results:Antibody blockade of TSP1 or morpholino suppression of CD47 dramatically enhanced survival of random tissue flaps. These responses correlated with increased blood vessel patency and tissue blood flow on vessel injection studies. NO-stimulated cGMP flux in Yucatan vascular smooth muscle cell was abrogated after antibody or morpholino treatment. Conclusion:Antibody ligation of TSP1 or antisense morpholino knock down of CD47 greatly increased tissue survival to ischemia. Given the similarity between porcine and human soft tissues these results suggest significant therapeutic potential for people.

Heparan Sulfate Modification of the Transmembrane Receptor CD47 Is Necessary for Inhibition of T Cell Receptor Signaling by Thrombospondin-1

Cell surface proteoglycans on T cells contribute to retroviral infection, binding of chemokines and other proteins, and are necessary for some T cell responses to the matricellular glycoprotein thrombospondin-1. The major cell surface proteoglycans expressed by primary T cells and Jurkat T cells have an apparent Mr > 200,000 and are modified with chondroitin sulfate and heparan sulfate chains. Thrombospondin-1 bound in a heparin-inhibitable manner to this proteoglycan and to a soluble form released into the medium. Based on mass spectrometry, knockdown, and immunochemical analyses, the proteoglycan contains two major core proteins as follows: amyloid precursor-like protein-2 (APLP2, apparent Mr 230,000) and CD47 (apparent Mr > 250,000). CD47 is a known thrombospondin-1 receptor but was not previously reported to be a proteoglycan. This proteoglycan isoform of CD47 is widely expressed on vascular cells. Mutagenesis identified glycosaminoglycan modification of CD47 at Ser64 and Ser79. Inhibition of T cell receptor signaling by thrombospondin-1 was lost in CD47-deficient T cells that express the proteoglycan isoform of APLP2, indicating that binding to APLP2 is not sufficient. Inhibition of CD69 induction was restored in CD47-deficient cells by re-expressing CD47 or an S79A mutant but not by the S64A mutant. Therefore, inhibition of T cell receptor signaling by thrombospondin-1 is mediated by CD47 and requires its modification at Ser64.

Measuring tissue-based biomarkers by immunochromatography coupled with reverse-phase lysate microarray

Purpose: There is a need for new technologies to study tissue-based biomarkers. The current gold standard, immunohistochemistry, is compromised by variability in tissue processing and observer bias. Reverse transcription-PCR (RT-PCR), immunocytochemistry, and reverse-phase lysate microarrays (RPM) are promising alternative technologies but have not yet been validated, or correlated, on the same patient-derived tissues. Furthermore, RPM is currently limited by time-consuming microdissection and low amounts of evaluable protein lysates. Experimental Design: Metastatic melanoma was surgically excised from 30 patients and macroscopically dissected from surrounding stroma. Each specimen was processed by formalin-fixation (immunohistochemistry), cytospin (immunocytochemistry), or disaggreagation and enrichment (RT-PCR and RPM). The latter protocol uses immunochromatography to remove hematopoetic-derived cells, thus enriching for melanoma cells. Each sample was measured for the expression of gp100 or MART-1 normalized to actin. Results: Immunochromatography coupled with RPM (I-RPM) is reproducible (r ≥ 0.70) and, for gp100, correlates strongly with immunohistochemistry and immunocytochemistry (r = 0.78 and 0.76, respectively) and moderately with transcript levels, measured by RT-PCR (r = 0.61). In contrast, for MART-1, I-RPM correlates strongly with transcript level (r = 0.78) but only moderately strong correlations are noted with immunohistochemistry and immunocytochemistry (r = 0.64 and 0.59, respectively). In general, transcript levels show only moderately strong correlations with immunohistochemistry and immunocytochemistry (r = 0.41-0.64). Conclusion: I-RPM is a promising technology for quantitative grading of tissue biomarkers; however, antigen-dependent correlations are noted.