Jeroen Hagendoorn

Imaging Steps of Lymphatic Metastasis Reveals That Vascular Endothelial Growth Factor-C Increases Metastasis by Increasing Delivery of Cancer Cells to Lymph Nodes: Therapeutic Implications

Preclinical and clinical studies positively correlate the expression of vascular endothelial growth factor (VEGF)-C in tumors and the incidence of lymph node metastases. However, how VEGF-C regulates individual steps in the transport of tumor cells from the primary tumor to the draining lymph nodes is poorly understood. Here, we image and quantify these steps in tumors growing in the tip of the mouse ear using intravital microscopy of the draining lymphatic vessels and lymph node, which receives spontaneously shed tumor cells. We show that VEGF-C overexpression in cancer cells induces hyperplasia in peritumor lymphatic vessels and increases the volumetric flow rate in lymphatics at the base of the ear by 40%. The increases in lymph flow rate and peritumor lymphatic surface area enhance the rate of tumor cell delivery to lymph nodes, leading to a 200-fold increase in cancer cell accumulation in the lymph node and a 4-fold increase in lymph node metastasis. In our model, VEGF-C overexpression does not confer any survival or growth advantage on cancer cells. We also show that an anti-VEGF receptor (VEGFR)-3 antibody reduces both lymphatic hyperplasia and the delivery of tumor cells to the draining lymph node, leading to a reduction in lymph node metastasis. However, this treatment is unable to prevent the growth of tumor cells already seeded in lymph nodes. Collectively, our results indicate that VEGF-C facilitates lymphatic metastasis by increasing the delivery of cancer cells to lymph nodes and therapies directed against VEGF-C/VEGFR-3 signaling target the initial steps of lymphatic metastasis.

Peritumor lymphatics induced by vascular endothelial growth factor-C exhibit abnormal function

Vascular endothelial growth factor (VEGF)-C is known to induce hyperplasia in normal murine lymphatics and in peritumor lymphatics. Here, we examine the function of these hyperplastic peritumor lymphatics. Microlymphangiography of B16F10 melanomas growing in the murine dorsal skinfold chamber showed that the number of functional, draining lymphatics in the peritumor tissue of VEGF-C-overexpressing tumors was significantly greater than that in mock-transduced tumors (9.5 ± 1.0 versus 6.3 ± 0.4; n = 6; P < 0.05). Forty percent of functional lymphatics associated with VEGF-C-overexpressing tumors contained proliferating lymphatic endothelial cells. Surprisingly, these new, functional lymphatic vessels displayed a retrograde draining pattern, which indicates possible dysfunction of the intraluminal valves of these vessels.

Onset of Abnormal Blood and Lymphatic Vessel Function and Interstitial Hypertension in Early Stages of Carcinogenesis

Recent improvements in diagnostic methods have opened avenues for detection and treatment of (pre)malignant lesions at early stages. However, due to the lack of spontaneous tumor models that both mimic human carcinogenesis and allow direct optical imaging of the vasculature, little is known about the function of blood and lymphatic vessels during the early stages of cancer development. Here, we used a spontaneous carcinogenesis model in the skin of DNA polymerase eta-deficient mice and found that interstitial fluid pressure was already elevated in the hyperplastic/dysplastic stage. This was accompanied by angiogenic blood vasculature that exhibited altered permeability, vessel compression, and decreased alpha-smooth muscle actin-positive perivascular cell coverage. In addition, the lymphatic vessels in hyperplastic/dysplastic lesions were partly compressed and nonfunctional. These novel insights may aid early detection and treatment strategies for cancer.

Endothelial Nitric Oxide Synthase Mediates Lymphangiogenesis and Lymphatic Metastasis

Lymphatic metastasis is a critical determinant of cancer prognosis. Recently, several lymphangiogenic molecules such as vascular endothelial growth factor (VEGF)-C and VEGF-D were identified. However, the mechanistic understanding of lymphatic metastasis is still in infancy. Nitric oxide (NO) plays a crucial role in regulating blood vessel growth and function as well as lymphatic vessel function. NO synthase (NOS) expression correlates with lymphatic metastasis. However, causal relationship between NOS and lymphatic metastasis has not been documented. To this end, we first show that both VEGF receptor-2 and VEGF receptor-3 stimulation activate eNOS in lymphatic endothelial cells and that NO donors induce proliferation and/or survival of cultured lymphatic endothelial cells in a dose-dependent manner. We find that an NOS inhibitor, L-NMMA, blocked regeneration of lymphatic vessels. Using intravital microscopy that allows us to visualize the steps of lymphatic metastasis, we show that genetic deletion of eNOS as well as NOS blockade attenuates peritumor lymphatic hyperplasia of VEGF-C-overexpressing T241 fibrosarcomas and decreases the delivery of metastatic tumor cells to the draining lymph nodes. Genetic deletion of eNOS in the host also leads to a decrease in T241 tumor cell dissemination to the lymph nodes and macroscopic lymph node metastasis of B16F10 melanoma. These findings indicate that eNOS mediates VEGF-C-induced lymphangiogenesis and, consequently, plays a critical role in lymphatic metastasis. Our findings explain the correlation between NOS and lymphatic metastasis seen in a number of human tumors and open the door for potential therapies exploiting NO signaling to treat diseases of the lymphatic system.

Endothelial Nitric Oxide Synthase Regulates Microlymphatic Flow via Collecting Lymphatics

Functional interactions between the initial and collecting lymphatics, as well as the molecular players involved, remain elusive. In this study, we assessed the influence of nitric oxide (NO) on lymphatic fluid velocity and flow, using a mouse tail model that permits intravital microscopy and microlymphangiography. We found that NO synthase (NOS) inhibition decreased lymphatic fluid velocity in the initial lymphatics, without any effect on their morphology. Using the same model, we found a similar effect in eNOS−/− mice and in mice treated with a selective endothelial NOS (eNOS) inhibitor. Next, we uncoupled the superficial initial lymphatics from the deeper collecting lymphatics by ligating the latter and found that lymphatic fluid velocity in NOS-inhibited mice became equal to that in control animals. Surprisingly, lymphatic fluid velocity was significantly increased after ligating the collecting lymphatics, and there was a concomitant increase in injection rate and mean lymphatic vessel diameter. Our results provide the first in vivo evidence that eNOS affects function of the whole microlymphatic system and that it is regulated via the collecting lymphatics.

Platelet-derived growth factor receptor-β in Gorham's disease

Background A 17-year-old male presented with pain in his lower-left chest. He had no significant medical history and was previously in good health. He had a fractured ninth left anterior rib and the tenth, eleventh and twelfth ribs were absent, which was thought to be a congenital anomaly. Several months later, he presented again with back pain, an enlarging mass in the lower-left chest wall, erosion of the lateral pedicles of the lower thoracic vertebrae and pleural effusion.Investigations Physical examination, chest X-ray, MRI of the spine, incisional biopsy, serial CT imaging of the hemithorax, immunohistochemistry, flow cytometry, and enzyme-linked immunosorbent assays.Diagnosis Gorhams lymphangiomatosis with expression of platelet-derived growth factor receptor-β and elevated circulating platelet-derived growth factor-BB.Management Spine stabilization, thalidomide, celecoxib, interferon-α2b, pamidronate, zoledronate, thoracotomy, pleurectomy, talc pleurodesis, and imatinib mesylate.

Adverse effects of the antiangiogenic agent angiostatin on the healing of experimental colonic anastomoses.

BackgroundAntiangiogenic cancer therapy is likely to be administered long term for sustained suppression of tumor outgrowth. Surgeons will encounter more patients undergoing such therapy. Therefore, it is essential to know the effects of antiangiogenic agents on physiological angiogenesis, as occurs during the healing of colonic anastomoses.MethodsAngiostatin was generated from human plasma and administered continuously. In 38 mice, the right colon was anastomosed after transection: group 1 (n=13), anstomotic healing under angiostatin treatment from surgery until death (day 7); group 2 (n=13), phosphate-buffered saline controls. For healing on discontinuation of treatment, group 3 (n=6) received angiostatin treatment preceding surgery during 4 days; group 4 (n=6) included controls. On day 7, all mice were inspected for signs of anastomotic leakage. Bursting pressure measurements were performed to test anastomotic strength. Neovascularization was assessed semiquantitatively by immunohistochemistry.ResultsMice treated with angiostatin postoperatively showed significantly more signs of leakage, more adhesions and peritonitis. One mouse died on day, 5. Five mice had paralytical ileus. The bursting pressure in group 1 was 135±20 mm Hg, versus 175±12 mm Hg in group 2 (mean ±SEM). Significantly fewer new vessels were found surrounding the anastomosis in the treated group (6.6±.9) versus controls (16±1.6). All controls, as well as those animals treated with angiostatin only until surgery (group 3), displayed normal healing and showed no signs of peritonitis or ileus.ConclusionsAngiostatin impairs anastomotic healing in mice. However, on discontinuation of antiangiogenic therapy, normal anastomotic healing is promptly restored.

Lack of lymphatic vessel phenotype in LYVE-1/CD44 double knockout mice.

Lymphatic vessels play a key role in maintaining tissue‐fluid homeostasis, immune surveillance and metastasis. The hyaluronan receptor, LYVE‐1, is widely used as a molecular marker for adult and embryonic lymphatic endothelium, but its physiological functions have not yet been established in vivo. In agreement with a recent report, LYVE‐1−/− mice, which are healthy and fertile, do not display any defects related to congenital abnormalities of the lymphatic system. One hypothesis for the absence of a phenotype in LYVE‐1 null mice is that other hyaluronan receptors, such as CD44, may compensate for LYVE‐1. To test this hypothesis, we created LYVE‐1/CD44 double knockout mice with appropriate littermate controls. Lymphatic vessel structure and function, as determined by histological methods and intravital microscopy, show that LYVE‐1−/−, CD44−/− and LYVE‐1−/−/CD44−/− mice are indistinguishable from wild‐type mice under normal conditions. Furthermore, resolution of carrageenan‐induced paw edema is comparable in all genotypes. However, LYVE‐1−/−/CD44−/− mice exhibit increased edema formation in a carrageenan‐induced paw inflammation model compared to wild‐type mice, but not to LYVE−/− or CD44−/− mice. These data suggest that LYVE‐1 and CD44 are not required for the formation or function of lymphatics, but do not rule out a role for LYVE‐1 in inflammation. J. Cell. Physiol. 219: 430–437, 2009.

Health‐related quality of life after pancreatic resection for malignancy

Health‐related quality of life (QoL) is of major importance in pancreatic cancer, owing to the limited life expectation. The aim of this prospective longitudinal study was to describe QoL in patients undergoing resection for pancreatic or periampullary malignancy.

Mesenterical Lymphangiomatosis Causing Volvulus and Intestinal Obstruction

Lymphangiomas are benign tumors consisting of lymphatic vasculature that generally occur in the skin and soft tissues. Rarely, lymphangiomas occur in the gastrointestinal tract. Here, we report a case of a 13-year-old girl presenting with an intestinal obstruction. Upon laparotomy, multiple cystic masses in the mesentery causing a volvulus were resected and histologically identified as multiple lymphangiomas, or lymphangiomatosis. Mesenteric lymphangioma is a rare entity, but should be considered as cause of bowel obstruction without other known abdominal disease. As the etiology of lymphangiomas remains elusive, further research is directed at unravelling the mechanistic and molecular factors contributing to this disease.