Clinical Focus

  • Hematopathology
  • Anatomic Pathology

Academic Appointments

Professional Education

  • Fellowship, Stanford University Hospital and Clinic - Anatomic Pathology, Hematopathology (2008)
  • Fellowship:Stanford University Hospital and Clinic - Anatomic Pathology (2007) CA
  • Residency:Stanford University Hospital and Clinic - Anatomic Pathology (2006) CA
  • Residency:Stanford University Hospital and Clinic - Anatomic Pathology (2005) CA
  • Medical Education:Yale University School of Medicine (2003) CT
  • Board Certification: Hematopathology, American Board of Pathology (2008)
  • Board Certification: Anatomic Pathology, American Board of Pathology (2006)
  • MD, Yale University, Medicine (2003)
  • PhD, Yale University, Experimental Pathology (2003)
  • BA, U. of California, Berkeley, Biochemistry (1996)

Research & Scholarship

Current Research and Scholarly Interests

I have research interests in the interaction of hematolymphoid neoplasia with the microenvironment. For example, I use a combination of immunohistochemistry, immunofluorescence and image analysis techniques to evaluate the mesenchymal stromal cell compartment in myelodysplastic syndrome (pre-leukemic bone marrow failure disorder). I also have interests in lymphoma vasculature and the tropism of lymphoma for specific types of vasculature.


2014-15 Courses

Graduate and Fellowship Programs

  • Hematopathology (Fellowship Program)


Journal Articles

  • Mesenchymal Stromal Cell Density Is Increased in Higher Grade Myelodysplastic Syndromes and Independently Predicts Survival AMERICAN JOURNAL OF CLINICAL PATHOLOGY Johnson, R. C., Kurzer, J. H., Greenberg, P. L., Gratzinger, D. 2014; 142 (6): 795-802
  • Intralymphatic Cutaneous Anaplastic Large Cell Lymphoma/Lymphomatoid Papulosis: Expanding the Spectrum of CD30-positive Lymphoproliferative Disorders. American journal of surgical pathology Samols, M. A., Su, A., Ra, S., Cappel, M. A., Louissant, A., Knudson, R. A., Ketterling, R. P., Said, J., Binder, S., Harris, N. L., Feldman, A. L., Kim, J., Kim, Y. H., Gratzinger, D. 2014; 38 (9): 1203-1211


    Intravascular large B-cell lymphomas and EBV NK/T-cell lymphomas commonly follow an aggressive clinical course. We recently reported an entirely intravascular anaplastic large cell lymphoma (ALCL) in the skin with a surprisingly indolent clinical course; interestingly, this lymphoma involved the lymphatic rather than the blood vasculature. We hypothesized that intravascular skin-limited ALCL is distinct from aggressive systemic intravascular lymphomas in its intralymphatic localization and clinical course. We now describe 18 cases of cutaneous intravascular large cell lymphoproliferations from 4 institutions. All 12 intravascular large T-cell lesions were intralymphatic; the majority (9) were CD30 T-cell lymphoproliferative disorders (TLPDs), 5 further classified as intravascular ALK ALCL. One ALK ALCL and 2 benign microscopic intravascular T-cell proliferations were also intralymphatic. A single case of otherwise typical cutaneous follicle center lymphoma contained intralymphatic centroblasts. The clinical and pathologic characteristics of the CD30 TLPDs were similar to those of their extravascular counterparts, including extralymphatic dermal involvement in a subset, DUSP22-IRF4 translocations in half of tested ALK ALCLs, and associated mycosis fungoides in 1; most were skin-limited at baseline and remained so at relapse. All 5 cases of intravascular large B-cell lymphoma involved the blood vasculature and behaved in a clinically aggressive manner; the ALK ALCL, although intralymphatic, was systemic and clinically aggressive. We propose that cutaneous ALK ALCL and related CD30 ALK TLPDs involving the lymphatics are part of an expanding spectrum of CD30 TLPDs. The identification of intralymphatic as distinct from blood vascular localization may provide critical prognostic and therapeutic information.

    View details for DOI 10.1097/PAS.0000000000000217

    View details for PubMedID 24805854

  • Vascular endothelial growth factor: the salt in the Hodgkin cytokine stew? Leukemia & lymphoma Gratzinger, D. 2014; 55 (3): 474-475

    View details for DOI 10.3109/10428194.2013.818144

    View details for PubMedID 23795806

  • Intravascular ALK-negative Anaplastic Large Cell Lymphoma With Localized Cutaneous Involvement and an Indolent Clinical Course Toward Recognition of a Distinct Clinicopathologic Entity AMERICAN JOURNAL OF SURGICAL PATHOLOGY Metcalf, R. A., Bashey, S., Wysong, A., Kim, J., Kim, Y. H., Gratzinger, D. 2013; 37 (4): 617-623


    Intravascular large T-cell or NK-cell lymphomas rarely present with cutaneous involvement. Intravascular cytotoxic T or NK lymphomas presenting in the skin (cIT/NKL) are often EBV, and reported cases follow a highly aggressive clinical course. Intravascular anaplastic large cell lymphoma (ALCL) by contrast is extraordinarily rare and, when it presents in the skin, raises the question of aggressive clinical behavior in the manner of cIT/NKL versus indolent clinical behavior in the manner of primary cutaneous ALCL. Here we describe a case of localized cutaneous intravascular anaplastic lymphoma kinase-negative ALCL (cIALCL) with a very indolent clinical course. The patient experienced a single cutaneous relapse and remains alive without disease 4 years after diagnosis. Review of the literature reveals multiple clinicopathologic differences between cIALCL and cIT/NKL: distribution (cIALCL, single skin region, P=0.021, Fisher exact test); histology (cIALCL, cohesive with necrosis, P=0.005); immunophenotype (cIALCL, strongly CD30, P=0.021; cIT/NKL, CD56 and/or EBV, P=0.003); and indolent clinical behavior with a trend toward better overall survival (P=0.067, Kaplan-Meier survival analysis). Our index case of cIALCL and 1 other tested case were immunohistochemically confirmed to be intralymphatic (contained within D2-40+vessels) as compared with the blood vessel localization of cIT/NKL. Recognition of cIALCLs as a distinct clinicopathologic entity, and in particular their distinction from aggressive, usually EBV cIT/NKLs, may be possible on the basis of a combination of clinicopathologic criteria, allowing for localized therapy in a subset of patients.

    View details for DOI 10.1097/PAS.0b013e318280aa9c

    View details for Web of Science ID 000316184000019

  • Update on Myelodysplastic Syndromes Classification and Prognosis Surgical Pathology Clinics Gratzinger, D., Greenberg, P. L. 2013; 6 (4): 693-728
  • Distinctive contact between CD34+ hematopoietic progenitors and CXCL12+ CD271+ mesenchymal stromal cells in benign and myelodysplastic bone marrow LABORATORY INVESTIGATION Flores-Figueroa, E., Varma, S., Montgomery, K., Greenberg, P. L., Gratzinger, D. 2012; 92 (9): 1330-1341


    Mesenchymal stromal cells (MSCs) support hematopoiesis and are cytogenetically and functionally abnormal in myelodysplastic syndrome (MDS), implying a possible pathophysiologic role in MDS and potential utility as a diagnostic or risk-stratifying tool. We have analyzed putative MSC markers and their relationship to CD34+ hematopoietic stem/progenitor cells (HSPCs) within intact human bone marrow in paraffin-embedded bone marrow core biopsies of benign, MDS and leukemic (AML) marrows using tissue microarrays to facilitate scanning, image analysis and quantitation. We found that CD271+, ALP+ MSCs formed an extensive branching perivascular, periosteal and parenchymal network. Nestin was brightly positive in capillary/arteriolar endothelium and occasional subendothelial cells, whereas CD146 was most brightly expressed in SMA+ vascular smooth muscle/pericytes. CD271+ MSCs were distinct by double immunofluorescence from CD163+ macrophages and were in close contact with but distinct from brightly nestin+ and from brightly CD146+ vascular elements. Double immunofluorescence revealed an intimate spatial relationship between CD34+ HSPCs and CD271+ MSCs; remarkably, 86% of CD34+ HSPCs were in direct contact with CD271+ MSCs across benign, MDS and AML marrows, predominantly in a perivascular distribution. Expression of the intercrine chemokine CXCL12 was strong in the vasculature in both benign and neoplastic marrow, but was also present in extravascular parenchymal cells, particularly in MDS specimens. We identified these parenchymal cells as MSCs by ALP/CXCL12 and CD271/CXCL12 double immunofluorescence. The area covered by CXCL12+ ALP+ MSCs was significantly greater in MDS compared with benign and AML marrow (P=0.021, Kruskal-Wallis test). The preservation of direct CD271+ MSC/CD34+ HSPC contact across benign and neoplastic marrow suggests a physiologically important role for the CD271+ MSC/CD34+ HSPC relationship and possible abnormal exposure of CD34+ HSPCs to increased MSC CXCL12 expression in MDS.

    View details for DOI 10.1038/labinvest.2012.93

    View details for Web of Science ID 000308274600008

    View details for PubMedID 22710983

  • In situ vaccination against mycosis fungoides by intratumoral injection of a TLR9 agonist combined with radiation: a phase 1/2 study BLOOD Kim, Y. H., Gratzinger, D., Harrison, C., Brody, J. D., Czerwinski, D. K., Ai, W. Z., Morales, A., Abdulla, F., Xing, L., Navi, D., Tibshirani, R. J., Advani, R. H., Lingala, B., Shah, S., Hoppe, R. T., Levy, R. 2012; 119 (2): 355-363


    We have developed and previously reported on a therapeutic vaccination strategy for indolent B-cell lymphoma that combines local radiation to enhance tumor immunogenicity with the injection into the tumor of a TLR9 agonist. As a result, antitumor CD8(+) T cells are induced, and systemic tumor regression was documented. Because the vaccination occurs in situ, there is no need to manufacture a vaccine product. We have now explored this strategy in a second disease: mycosis fungoides (MF). We treated 15 patients. Clinical responses were assessed at the distant, untreated sites as a measure of systemic antitumor activity. Five clinically meaningful responses were observed. The procedure was well tolerated and adverse effects consisted mostly of mild and transient injection site or flu-like symptoms. The immunized sites showed a significant reduction of CD25(+), Foxp3(+) T cells that could be either MF cells or tissue regulatory T cells and a similar reduction in S100(+), CD1a(+) dendritic cells. There was a trend toward greater reduction of CD25(+) T cells and skin dendritic cells in clinical responders versus nonresponders. Our in situ vaccination strategy is feasible also in MF and the clinical responses that occurred in a subset of patients warrant further study with modifications to augment these therapeutic effects. This study is registered at as NCT00226993.

    View details for DOI 10.1182/blood-2011-05-355222

    View details for Web of Science ID 000299268900012

    View details for PubMedID 22045986

  • CD81 protein is expressed at high levels in normal germinal center B cells and in subtypes of human lymphomas HUMAN PATHOLOGY Luo, R. F., Zhao, S., Tibshirani, R., Myklebust, J. H., Sanyal, M., Fernandez, R., Gratzinger, D., Marinelli, R. J., Lu, Z. S., Wong, A., Levy, R., Levy, S., Natkunam, Y. 2010; 41 (2): 271-280


    CD81 is a tetraspanin cell surface protein that regulates CD19 expression in B lymphocytes and enables hepatitis C virus infection of human cells. Immunohistologic analysis in normal hematopoietic tissue showed strong staining for CD81 in normal germinal center B cells, a cell type in which its increased expression has not been previously recognized. High-dimensional flow cytometry analysis of normal hematopoietic tissue confirmed that among B- and T-cell subsets, germinal center B cells showed the highest level of CD81 expression. In more than 800 neoplastic tissue samples, its expression was also found in most non-Hodgkin lymphomas. Staining for CD81 was rarely seen in multiple myeloma, Hodgkin lymphoma, or myeloid leukemia. In hierarchical cluster analysis of diffuse large B-cell lymphoma, staining for CD81 was most similar to other germinal center B cell-associated markers, particularly LMO2. By flow cytometry, CD81 was expressed in diffuse large B-cell lymphoma cells independent of the presence or absence of CD10, another germinal center B-cell marker. The detection of CD81 in routine biopsy samples and its differential expression in lymphoma subtypes, particularly diffuse large B-cell lymphoma, warrant further study to assess CD81 expression and its role in the risk stratification of patients with diffuse large B-cell lymphoma.

    View details for DOI 10.1016/j.humpath.2009.07.022

    View details for Web of Science ID 000276493600015

    View details for PubMedID 20004001

  • Lymphoma cell VEGFR2 expression detected by immunohistochemistry predicts poor overall survival in diffuse large B cell lymphoma treated with immunochemotherapy (R-CHOP) BRITISH JOURNAL OF HAEMATOLOGY Gratzinger, D., Advani, R., Zhao, S., Talreja, N., Tibshirani, R. J., Shyam, R., Horning, S., Sehn, L. H., Farinha, P., Briones, J., Lossos, I. S., Gascoyne, R. D., Natkunam, Y. 2010; 148 (2): 235-244


    Diffuse large B cell lymphoma (DLBCL) is clinically and biologically heterogeneous. In most cases of DLBCL, lymphoma cells co-express vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2, suggesting autocrine in addition to angiogenic effects. We enumerated microvessel density and scored lymphoma cell expression of VEGF, VEGFR1, VEGFR2 and phosphorylated VEGFR2 in 162 de novo DLBCL patients treated with R-CHOP (rituximab, cyclophosphamide, vincristine, doxorubicin and prednisone)-like regimens. VEGFR2 expression correlated with shorter overall survival (OS) independent of International Prognostic Index (IPI) (P = 0.0028). Phosphorylated VEGFR2 (detected in 13% of cases) correlated with shorter progression-free survival (PFS, P = 0.044) and trended toward shorter OS on univariate analysis. VEGFR1 was not predictive of survival on univariate analysis, but it did correlate with better OS on multivariate analysis with VEGF, VEGFR2 and IPI (P = 0.036); in patients with weak VEGFR2, lack of VEGFR1 coexpression was significantly correlated with poor OS independent of IPI (P = 0.01). These results are concordant with our prior finding of an association of VEGFR1 with longer OS in DLBCL treated with chemotherapy alone. We postulate that VEGFR1 may oppose autocrine VEGFR2 signalling in DLBCL by competing for VEGF binding. In contrast to our prior results with chemotherapy alone, microvessel density was not prognostic of PFS or OS with R-CHOP-like therapy.

    View details for DOI 10.1111/j.1365-2141.2009.07942.x

    View details for Web of Science ID 000272884100006

    View details for PubMedID 19821819

  • The Transcription Factor LMO2 Is a Robust Marker of Vascular Endothelium and Vascular Neoplasms and Selected Other Entities AMERICAN JOURNAL OF CLINICAL PATHOLOGY Gratzinger, D., Zhao, S., West, R., Rouse, R. V., Vogel, H., Gil, E. C., Levy, R., Lossos, I. S., Natkunam, Y. 2009; 131 (2): 264-278


    The transcription factor LMO2 is involved in vascular and hematopoietic development and hematolymphoid neoplasia. We have demonstrated that LMO2 is expressed nearly ubiquitously in native and neoplastic vasculature, including lymphatics. LMO2 reactivity is otherwise virtually absent in nonhematolymphoid tissues except in breast myoepithelium, prostatic basal cells, and secretory phase endometrial glands. Vasculature is LMO2- in adult and fetal heart, brain of older adults, hepatic sinusoids, and hepatocellular carcinoma. LMO2 is uniformly expressed in benign vascular and lymphatic neoplasms and in most malignant vascular neoplasms with the exception of epithelioid vascular neoplasms of pleura and bone. Among nonvascular neoplasms, LMO2 reactivity is present in giant cell tumor of tendon sheath, juvenile xanthogranuloma, a subset of gastrointestinal stromal tumors, small round blue cell tumors, and myoepithelial-derived neoplasms. The restricted expression pattern, nuclear localization, and crisp staining of LMO2 in paraffin blocks make it an attractive candidate for the diagnostic immunohistochemistry laboratory.

    View details for DOI 10.1309/AJCP5FP3NAXAXRJE

    View details for Web of Science ID 000262540200013

    View details for PubMedID 19141387

  • VEGF-C: putting the 'lymph' back in lymphoma? LEUKEMIA & LYMPHOMA Gratzinger, D. 2009; 50 (3): 311-312

    View details for DOI 10.1080/10428190902763525

    View details for Web of Science ID 000265361100002

    View details for PubMedID 19347719

  • Ameloblastoma, calcifying epithelial odontogenic tumor, and glandular odontogenic cyst show a distinctive immunophenotype with some myoepithelial antigen expression JOURNAL OF ORAL PATHOLOGY & MEDICINE Gratzinger, D., Salama, M. E., Poh, C. F., Rouse, R. V. 2008; 37 (3): 177-184


    Odontogenic neoplasms have some morphologic overlap with salivary gland neoplasms, many of which show myoepithelial differentiation. In the 1980s, an ultrastructural study identified a population of myoepithelial-like cells in calcifying epithelial odontogenic tumor. Myoepithelial derived tumors have since been shown to have distinct immunohistochemical profiles.We examined a series of odontogenic neoplasms, including 11 ameloblastomas, four calcifying epithelial odontogenic tumors, five glandular odontogenic cysts (GOCs), and five keratocystic odontogenic tumors with a panel of myoepithelial-associated immunohistochemical stains. We also assessed representative control examples of oral mucosa, odontogenic rests, and dentigerous cysts.All of the neoplastic and non-neoplastic oral epithelium-derived entities share a p63-positive, high molecular weight cytokeratin (CK5/6)-positive immunophenotype. Calponin reactivity was at least focally present in two of four calcifying epithelial odontogenic tumors, three of five GOCs, and 10 of 11 ameloblastomas; the sole completely non-reactive ameloblastoma represents a lung metastasis. One case of calcifying epithelial odontogenic tumor was focally positive for glial fibrillary acidic protein. However, other more definitive markers of myoepithelial differentiation, including S-100 and smooth muscle actin, were negative. Two of three calcifying epithelial odontogenic tumors and five of five GOCs were also positive for a low molecular weight cytokeratin (CK7).Ameloblastomas, GOCs, and calcifying epithelial odontogenic tumors show a distinctive immunophenotype which overlaps with that of myoepithelial-derived salivary gland neoplasms but does not provide definitive support for myoepithelial differentiation.

    View details for DOI 10.1111/j.1600-0714.2007.00613.x

    View details for Web of Science ID 000252812000008

    View details for PubMedID 18251942

  • LMO2 protein expression predicts survival in patients with diffuse large B-Cell lymphoma treated with anthracycline-based chemotherapy with and without rituximab JOURNAL OF CLINICAL ONCOLOGY Natkunam, Y., Farinha, P., Hsi, E. D., Hans, C. P., Tibshirani, R., Sehn, L. H., Connors, J. M., Gratzinger, D., Rosado, M., Zhao, S., Pohlman, B., Wongchaowart, N., Bast, M., Avigdor, A., Schiby, G., Nagler, A., Byrne, G. E., Levy, R., Gascoyne, R. D., Lossos, I. S. 2008; 26 (3): 447-454


    The heterogeneity of diffuse large B-cell lymphoma (DLBCL) has prompted the search for new markers that can accurately separate prognostic risk groups. We previously showed in a multivariate model that LMO2 mRNA was a strong predictor of superior outcome in DLBCL patients. Here, we tested the prognostic impact of LMO2 protein expression in DLBCL patients treated with anthracycline-based chemotherapy with or without rituximab.DLBCL patients treated with anthracycline-based chemotherapy alone (263 patients) or with the addition of rituximab (80 patients) were studied using immunohistochemistry for LMO2 on tissue microarrays of original biopsies. Staining results were correlated with outcome.In anthracycline-treated patients, LMO2 protein expression was significantly correlated with improved overall survival (OS) and progression-free survival (PFS) in univariate analyses (OS, P = .018; PFS, P = .010) and was a significant predictor independent of the clinical International Prognostic Index (IPI) in multivariate analysis. Similarly, in patients treated with the combination of anthracycline-containing regimens and rituximab, LMO2 protein expression was also significantly correlated with improved OS and PFS (OS, P = .005; PFS, P = .009) and was a significant predictor independent of the IPI in multivariate analysis.We conclude that LMO2 protein expression is a prognostic marker in DLBCL patients treated with anthracycline-based regimens alone or in combination with rituximab. After further validation, immunohistologic analysis of LMO2 protein expression may become a practical assay for newly diagnosed DLBCL patients to optimize their clinical management.

    View details for DOI 10.1200/JCO.2007.13.0690

    View details for Web of Science ID 000254177200020

    View details for PubMedID 18086797

  • Prognostic significance of VEGF, VEGF receptors, and microvessel density in diffuse large B cell lymphoma treated with anthracycline-based chemotherapy LABORATORY INVESTIGATION Gratzinger, D., Zhao, S., Tibshirani, R. J., Hsi, E. D., Hans, C. P., Pohlman, B., Bast, M., Avigdor, A., Schiby, G., Nagler, A., Byrne, G. E., Lossos, I. S., Natkunam, Y. 2008; 88 (1): 38-47


    Vascular endothelial growth factor-mediated signaling has at least two potential roles in diffuse large B cell lymphoma: potentiation of angiogenesis, and potentiation of lymphoma cell proliferation and/or survival induced by autocrine vascular endothelial growth factor receptor-mediated signaling. We have recently shown that diffuse large B cell lymphomas expressing high levels of vascular endothelial growth factor protein also express high levels of vascular endothelial growth factor receptor-1 and vascular endothelial growth factor receptor-2. We have now assessed a larger multi-institutional cohort of patients with de novo diffuse large B cell lymphoma treated with anthracycline-based therapy to address whether tumor vascularity, or expression of vascular endothelial growth factor protein and its receptors, contribute to patient outcomes. Our results show that increased tumor vascularity is associated with poor overall survival (P=0.047), and is independent of the international prognostic index. High expression of vascular endothelial growth factor receptor-1 by lymphoma cells by contrast is associated with improved overall survival (P=0.044). The combination of high vascular endothelial growth factor and vascular endothelial growth factor receptor-1 protein expression by lymphoma cells identifies a subgroup of patients with improved overall (P=0.003) and progression-free (P=0.026) survival; these findings are also independent of the international prognostic index. The prognostic significance of overexpression of this ligand-receptor pair suggests that autocrine signaling via vascular endothelial growth factor receptor-1 may represent a survival or proliferation pathway in diffuse large B cell lymphoma. Dependence on autocrine vascular endothelial growth factor receptor-1-mediated signaling may render a subset of diffuse large B-cell lymphomas susceptible to anthracycline-based therapy.

    View details for DOI 10.1038/labinvest.3700697

    View details for Web of Science ID 000251820600004

    View details for PubMedID 17998899

  • Microvessel density and expression of vascular endothelial growth factor and its receptors in diffuse large B-cell lymphoma subtypes AMERICAN JOURNAL OF PATHOLOGY Gratzinger, D., Zhao, S., Marinelli, R. J., Kapp, A. V., Tibshirani, R. J., Hammer, A. S., Hamilton-Dutoit, S., Natkunam, Y. 2007; 170 (4): 1362-1369


    Angiogenesis is known to play a major role in neoplasia, including hematolymphoid neoplasia. We assessed the relationships among angiogenesis and expression of vascular endothelial growth factor and its receptors in the context of clinically and biologically relevant subtypes of diffuse large B-cell lymphoma using immunohistochemical evaluation of tissue microarrays. We found that diffuse large B-cell lymphoma specimens showing higher local vascular endothelial growth factor expression showed correspondingly higher microvessel density, implying that lymphoma cells induce local tumor angiogenesis. In addition, local vascular endothelial growth factor expression was higher in those specimens showing higher expression of the receptors of the growth factor, suggesting an autocrine growth-promoting feedback loop. The germinal center-like and nongerminal center-like subtypes of diffuse large B-cell lymphoma were biologically and prognostically distinct. Interestingly, only in the more clinically aggressive nongerminal center-like subtype were microvessel densities significantly higher in specimens showing higher vascular endothelial growth factor expression; the same was true for the finding of higher vascular endothelial growth factor receptor-1 expression in conjunction with higher vascular endothelial growth factor expression. These differences may have important implications for the responsiveness of the two diffuse large B-cell lymphoma subtypes to anti-vascular endothelial growth factor and anti-angiogenic therapies.

    View details for DOI 10.2353/ajpath.2007.060901

    View details for Web of Science ID 000245233000022

    View details for PubMedID 17392174

  • Identification of the regions of PECAM-1 involved in beta- and gamma-catenin associations BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Biswas, P., Zhang, J., Schoenfeld, J. D., Schoenfeld, D., Gratzinger, D., Canosa, S., Madri, J. A. 2005; 329 (4): 1225-1233


    Platelet endothelial cell adhesion molecule-1 (PECAM-1) binds tyrosine-phosphorylated beta-catenin and modulates beta-catenin localization and sequestration. The biological significance of this interaction, while still unclear, it has been postulated to be involved in modulating adherens junction dynamics in response to perturbants [J. Clin. Invest. 109 (2002) 383]. Here we demonstrate that tyrosine-phosphorylated beta-catenin, and to a lesser extent unphosphorylated beta-catenin, interact with a portion of the cytoplasmic domain of PECAM-1 encoded by exon 15. Using RT-PCR, we obtained products representing alternatively spliced PECAM-1 isoforms from mouse kidney total mRNA and generated PECAM-1-GST constructs expressing full length and naturally occurring alternatively spliced PECAM-1 variants. Co-precipitation assays revealed that the protein sequence encoded by exon 15 is necessary for beta-catenin binding. Transfections using deletion mutants confirmed the importance of the exon 15 sequence in this interaction. In contrast, gamma-catenin-PECAM-1 interactions are thought to be modulated by an as yet undefined PECAM-1 serine phosphorylation and appear to mediate dynamic PECAM-1 intermediate filament cytoskeletal interactions [J. Biol. Chem. 275 (2000) 21435]. Here we demonstrate that the PECAM-1-gamma-catenin interaction occurs via an exon 13-mediated process. GST-pull-down assays illustrated the importance of the exon 13 sequence in this interaction. Further, using site-directed mutagenesis of S(673) to C and D and S(669 and 670) to C, we confirmed the importance of S(673) and its phosphorylation state as a mediator of gamma-catenin-PECAM-1 binding. Our studies define the exons of the PECAM-1 cytoplasmic domain that is involved in mediating these PECAM-1-catenin family member interactions and will allow investigators to better define the biological functions resulting from these interactions.

    View details for DOI 10.1016/j.bbrc.2005.02.095

    View details for Web of Science ID 000227886300008

    View details for PubMedID 15766557

  • Platelet endothelial cell adhesion molecule-1 modulates endothelial cell motility through the small G-protein Rho FASEB JOURNAL Gratzinger, D., Canosa, S., Engelhardt, B., Madri, J. A. 2003; 17 (11): 1458-1469


    Platelet endothelial cell adhesion molecule-1 (PECAM-1), an immunoglobulin family vascular adhesion molecule, is involved in endothelial cell migration and angiogenesis (1, 2). We found that endothelial cells lacking PECAM-1 exhibit increased single cell motility and extension formation but poor wound healing migration, reminiscent of cells in which Rho activity has been suppressed by overexpressing a GTPase-activating protein (3). The ability of PECAM-1 to restore wound healing migration to PECAM-1-deficient cells was independent of its extracellular domain or signaling via its immunoreceptor tyrosine-based inhibitory motif. PECAM-1-deficient endothelial cells had a selective defect in RhoGTP loading, and inhibition of Rho activity mimicked the PECAM-1-deficient phenotype of increased chemokinetic single cell motility at the expense of coordinated wound healing migration. The wound healing advantage of PECAM-1-positive endothelial cells was not only Rho mediated but pertussis toxin inhibitable, characteristic of migration mediated by heterotrimeric G-protein-linked seven-transmembrane receptor signaling such as signaling in response to the serum sphingolipid sphingosine-1-phosphate (S1P) (4, 5). Indeed, we found that the wound healing defect of PECAM-1 null endothelial cells is minimized in sphingolipid-depleted media; moreover, PECAM-1 null endothelial cells fail to increase their migration in response to S1P. We have also found that PECAM-1 localizes to rafts and that in its absence heterotrimeric G-protein components are differentially recruited to rafts, providing a potential mechanism for PECAM-1-mediated coordination of S1P signaling. PECAM-1 may thus support the effective S1P/RhoGTP signaling required for wound healing endothelial migration by allowing for the spatially directed, coordinated activation of Galpha signaling pathways.

    View details for DOI 10.1096/fj.02-1040com

    View details for Web of Science ID 000185345100039

    View details for PubMedID 12890700

  • Elevated glucose inhibits VEGF-A-mediated endocardial cushion formation: modulation by PECAM-1 and MMP-2 JOURNAL OF CELL BIOLOGY Enciso, J. M., Gratzinger, D., Camenisch, T. D., Canosa, S., Pinter, E., Madri, J. A. 2003; 160 (4): 605-615


    Atrioventricular (AV) septal defects resulting from aberrant endocardial cushion (EC) formation are observed at increased rates in infants of diabetic mothers. EC formation occurs via an epithelial-mesenchymal transformation (EMT), involving transformation of endocardial cells into mesenchymal cells, migration, and invasion into extracellular matrix. Here, we report that elevated glucose inhibits EMT by reducing myocardial vascular endothelial growth factor A (VEGF-A). This effect is reversed with exogenous recombinant mouse VEGF-A165, whereas addition of soluble VEGF receptor-1 blocks EMT. We show that disruption of EMT is associated with persistence of platelet endothelial cell adhesion molecule-1 (PECAM-1) and decreased matrix metalloproteinase-2 (MMP-2) expression. These findings correlate with retention of a nontransformed endocardial sheet and lack of invasion. The MMP inhibitor GM6001 blocks invasion, whereas explants from PECAM-1 deficient mice exhibit MMP-2 induction and normal EMT in high glucose. PECAM-1-negative endothelial cells are highly motile and express more MMP-2 than do PECAM-1-positive endothelial cells. During EMT, loss of PECAM-1 similarly promotes single cell motility and MMP-2 expression. Our findings suggest that high glucose-induced inhibition of AV cushion morphogenesis results from decreased myocardial VEGF-A expression and is, in part, mediated by persistent endocardial cell PECAM-1 expression and failure to up-regulate MMP-2 expression.

    View details for DOI 10.1083/jcb.200209014

    View details for Web of Science ID 000181090200014

    View details for PubMedID 12591918

  • Platelet-endothelial cell adhesion molecule-1 modulates endothelial migration through its immunoreceptor tyrosine-based inhibitory motif BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Gratzinger, D., Barreuther, M., Madri, J. A. 2003; 301 (1): 243-249


    Coordinated migration of endothelial cells models the remodeling of existing endothelia as well as angiogenesis and vasculogenesis. Platelet-endothelial cell adhesion molecule-1, PECAM-1, a transmembrane endothelial adhesion protein, binds and activates the tyrosine phosphatase SHP-2 via phosphotyrosines 663 and 686. PECAM-1 phosphorylation and recruitment of SHP-2 are regulated by cell-cell and cell-substrate adhesion. We found that PECAM-1 is dephosphorylated on tyrosine 686 during endothelial migration, resulting in diffuse dispersal of PECAM-1 and SHP-2. Overexpression of native PECAM-1 slowed, and nonphosphorylatable PECAM-1 increased, endothelial migration, implying that the SHP-2-regulatory phosphotyrosines negatively regulate migration. Using differentially phosphorylated recombinant proteins we found that phosphotyrosine 686 preferentially mediates binding and 663 mediates activation of SHP-2 by PECAM-1. In PECAM-1-null endothelial cells, SHP-2 bound and dephosphorylated an alternative set of phosphoproteins and its distribution to the cytoskeletal fraction was significantly decreased. Tyrosine phosphorylation of beta-catenin and focal adhesion kinase was increased in endothelial cells overexpressing nonphosphorylatable PECAM-1. Thus homophilically engaged, tyrosine-phosphorylated PECAM-1 locally activates SHP-2 at cell-cell junctions; with disruption of the endothelial monolayer, selective dephosphorylation of PECAM-1 leads to redistribution of SHP-2 and pro-migratory changes in phosphorylation of cytoskeletal and focal contact components.

    View details for DOI 10.1016/S0006-291X(02)02982-0

    View details for Web of Science ID 000181052800040

    View details for PubMedID 12535670

  • Interpreting a medium-resolution model of tubulin: Comparison of zinc-sheet and microtubule structure JOURNAL OF MOLECULAR BIOLOGY Wolf, S. G., Nogales, E., Kikkawa, M., Gratzinger, D., Hirokawa, N., Downing, K. H. 1996; 262 (4): 485-501


    We previously used electron crystallography of zinc-induced two-dimensional crystalline sheets of tubulin to construct a medium-resolution three dimensional (3-D) reconstruction (at 6.5 A) of this protein. Here we present an improved model, and extend the interpretation to correlate it to microtubule structure. Secondary sequence predictions and projection density maps of subtilisin-cleaved tubulin provide information on the location of the C-terminal portion, which has been suggested to be involved in the binding of microtubule-associated proteins. The zinc-sheet tubulin model is compared to microtubules in two ways; comparison of electron diffraction from the zinc-sheets to electron diffraction from microtubules, and by docking the zinc-sheet protofilament 3-D model into a helical reconstruction from ice-embedded microtubules. By correlating the zinc-sheet protofilament to a reconstruction of axonemal protofilaments, we assigned polarity to the protofilament in our model. The polarity assignment together with our model for dimer boundaries and the assignment of alpha- and beta-monomers in our reconstruction, provides a microtubule model where the alpha-monomer crowns the plus- (or fast-growing) end of the microtubule and contact is made in the centrosome with gamma-tubulin via the beta-monomer.

    View details for Web of Science ID A1996VK74900009

    View details for PubMedID 8893858

Conference Proceedings

  • In Situ Vaccination with TLR9 Agonist Combined with Local Radiation In Mycosis Fungoides: Analysis of Phase I/II Study Kim, Y. H., Gratzinger, D., Harrison, C., Brody, J., Czerwinski, D., Xing, L., Morales, A., Ai, W., Abdulla, F., Navi, D., Tibshirani, R. J., Advani, R., Natkunam, Y., Hoppe, R. T., Levy, R. AMER SOC HEMATOLOGY. 2010: 130-130
  • Prognostic significance of vascular endothelial growth factor (VEGF), VEGF receptors (VEGFR), and vascularity in diffuse large B-cell lymphoma treated with immunochemotherapy (R-CHOP) Gratzinger, D., Advani, R., Zhao, S., Talreja, N., Tibshirani, R. J., Horning, S. J., Levy, R., Lossos, I. S., Gascoyne, R. D., Natkunam, Y. AMER SOC CLINICAL ONCOLOGY. 2009
  • CD81 Protein Is Expressed in Normal Germinal Center B-Cells and in Subtypes of Human Non-Hodgkin Lymphomas Luo, R. F., Zhao, S., Tibshirani, R., Lossos, I. S., Advani, R., Gratzinger, D., Wong, A., Talrega, N., Levy, R., Levy, S., Natkunam, Y. NATURE PUBLISHING GROUP. 2009: 275A-275A
  • Lymphoma-Expressed VEGF-a,VEGFR-1, VEGFR-2, and Microvessel Density Are Not Predictive of Overall Survival in Follicular Lymphoma. Gratzinger, D., Zhao, S., Ai, W., Tibshirani, R., Levy, R., Natkunam, Y. AMER SOC HEMATOLOGY. 2008: 1290-1290
  • Neither CD68+Nor CD163+Macrophages Are Associated with Decreased Survival in Follicular Lymphoma Gratzinger, D., Ai, W., Tibshirani, R., Levy, R., Natkunam, Y. AMER SOC HEMATOLOGY. 2008: 1284-1284
  • The transcription factor LMO2 is a robust marker of vascular endothelium and vascular neoplasms with rare exceptions Gratzinger, D., Zhao, S., Vogel, H., Gil, E. C., Levy, R., Lossos, I., Natkunam, Y. FEDERATION AMER SOC EXP BIOL. 2008
  • LMO2 protein expression predicts survival in patients with diffuse large B-cell lymphoma treated with anthracycline-based chemotherapy with or without rituximab Natkunam, Y., Farinha, P., Hsi, E. D., Hans, C. P., Tibshirani, R., Sehn, L. H., Connors, J. M., Gratzinger, D., Zhan, S., Pohlman, B., Nagler, A., Levy, R., Gascoyne, R. D., Lossos, I. S. NATURE PUBLISHING GROUP. 2008: 267A-267A

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