Lymphatic endothelial progenitor cells contribute to de novo lymphangiogenesis in human renal transplants Dontscho Kerjaschki 1 , Nicole Huttary 1 , Ingrid Raab 1 , Heinz Regele 1 , Katalin Bojarski-Nagy 1 , Gregor Bartel 1 , Stefan M Kro ¨ber 2 , Hildegard Greinix 3 , Agathe Rosenmaier 3 , Franz Karlhofer 4 , Nikolaus Wick 1 & Peter R Mazal 1 De novo lymphangiogenesis influences the course of different human diseases as diverse as chronic renal transplant rejection 1 and tumor metastasis 2,3 . The cellular mechanisms of lymphangiogenesis in human diseases are currently unknown, and could involve division of local preexisting endothelial cells or incorporation of circulating progenitors. We analyzed renal tissues of individuals with gender-mismatched transplants who had transplant rejection and high rates of overall lymphatic endothelial proliferation as well as massive chronic inflammation. Donor-derived cells were detected by in situ hybridization of the Y chromosome. We compared these tissues with biopsies of essentially normal skin and intestine, and two rare carcinomas with low rates of lymphatic endothelial proliferation that were derived from individuals with gender- mismatched bone marrow transplants. Here, we provide evidence for the participation of recipient-derived lymphatic progenitor cells in renal transplants. In contrast, lymphatic vessels of normal tissues and those around post-transplant carcinomas did not incorporate donor-derived progenitors. This indicates a stepwise mechanism of inflammation-associated de novo lymphangiogenesis, implying that potential lymphatic progenitor cells derive from the circulation, transmigrate through the connective tissue stroma, presumably in the form of macrophages, and finally incorporate into the growing lymphatic vessel. De novo development of blood vessels (‘adult vasculogenesis’) 4 is important for cancer growth 5 and transplant survival 1 . For example, circulating endothelial progenitor cells are integrated into tumor blood vessels both in humans and in animal models, and thus provide a potential therapeutic target, as well as a surrogate marker for antiangiogenic tumor therapy 6 . In contrast, nothing is known about the mechanisms of de novo lymphangiogenesis in human diseases. Therefore, we examined whether circulating lymphatic endothelial progenitors have any role in this process, similar to the case with blood vessels, or alternatively, lymphatic networks grow by division of local endothelial cells. For this purpose, we used human tissues from male renal transplant recipients with a female donor kidney, or tissue from female bone marrow recipients who received a graft from a male donor. We detected the nuclei of progenitor-derived lymphatic endothelial cells through colocalization of the transcription factor Prox-1 (ref. 7) by immunohistochemistry, and the Y chromosome by in situ hybridization 8 . We chose nephrectomy specimens of rejected kidney grafts (n ¼ 6) that showed inflammation-associated, extensive de novo lymphangiogenesis 1 (Fig. 1a,b), whereas in normal human kidney only few lymphatic vessels were localized in the adventitia of large to middle-sized arteries. We examined normal or minimally inflamed skin and intestinal tract biopsies from recipients after bone marrow transplantation (n ¼ 32) as controls that presumably reflect lymphatic endothelial cell turnover in normal tissues. We also identi- fied in a worldwide search a post–bone marrow transplant mammary carcinoma and a colorectal carcinoma in appropriately gender-mis- matched recipients, each of which showed an elaborate peritumoral lymphatic vasculature and desmoplasia. Collectively, the results indicate that in renal explants, 47 out of 1,005 (4.5%; range, 2.7–7%) Prox-1 + lymphatic endothelial nuclei contained a single Y chromosome, and were therefore derived from circulating progenitors of the host’s genotype (Fig. 1d–h). These Y chromosome + endothelial cells accounted for 12.9% of the 281 lymphatic vessels encountered, suggesting that these vessels serve as focal sites of de novo angiogenesis (Table 1). We did not observe Y chromosome + pericytes. Fusion of endothelial progenitors with preexisting endothelial cells was discounted as we were unable to detect more than two sex chromosomes by double localization of X and Y chromosomes as assessed by fluorescent in situ hybridization in a large number (n ¼ 7,135) of nuclei (Supplementary Table 1 online). In contrast, 746 Prox-1 + nuclei of lymphatic endothelial cells in skin and gastrointestinal biopsies of individuals who had undergone bone marrow transplant did not contain a Y chromosome. Also, all 97 peritumoral lymphatic vessels (Fig. 2a–d) and their 338 Prox-1 + nuclei were devoid of Y chromosome + lymphatic endothelial cells (Table 1). As we used paraffin sections that were 4 mm thick, it is Received 25 May 2005; accepted 8 November 2005; published online 15 January 2006; doi:10.1038/nm1340 1 Department of Pathology, Medical University of Vienna - Allgemeines Krankenhaus Wa ¨ hringer Gu ¨ rtel 18 - 20, A 1090 Vienna, Austria. 2 Department of Pathology, University of Tu ¨ bingen, Liebermeisterstrae 8, D 72076 Tu ¨ bingen Germany. 3 Austrian Bone Marrow Donor Registry, Florianigasse 38/12, A 1080 Vienna, Austria. 4 Department of Dermatology, Medical University of Vienna - Allgemeines Krankenhaus Wa ¨hringer Gu ¨ rtel 18 - 20, A 1090 Vienna, Austria. Correspondence should be addressed to D.K. (dontscho.kerjaschki@meduniwien.ac.at). 230 VOLUME 12 [ NUMBER 2 [ FEBRUARY 2006 NATURE MEDICINE LETTERS © 2006 Nature Publishing Group http://www.nature.com/naturemedicine