V.Schacht and M.I.Ramirez contributed equally to this workWithin the vascular system, the mucin-type transmembrane glycoprotein T1a/podoplanin is predominantly expressed by lymphatic endothelium, and recent studies have shown that it is regulated by the lymphatic-speci®c homeobox gene Prox1. In this study, we examined the role of T1a/podoplanin in vascular development and the effects of gene disruption in mice. T1a/podoplanin is ®rst expressed at around E11.0 in Prox1-positive lymphatic progenitor cells, with predominant localization in the luminal plasma membrane of lymphatic endothelial cells during later development. T1a/podoplanin ±/± mice die at birth due to respiratory failure and have defects in lymphatic, but not blood vessel pattern formation. These defects are associated with diminished lymphatic transport, congenital lymphedema and dilation of lymphatic vessels. T1a/podoplanin is also expressed in the basal epidermis of newborn wild-type mice, but gene disruption did not alter epidermal differentiation. Studies in cultured endothelial cells indicate that T1a/podoplanin promotes cell adhesion, migration and tube formation, whereas small interfering RNA-mediated inhibition of T1a/podoplanin expression decreased lymphatic endothelial cell adhesion. These data identify T1a/podoplanin as a novel critical player that regulates different key aspects of lymphatic vasculature formation.

The mucin-type glycoprotein podoplanin is specifically expressed by lymphatic but not blood vascular endothelial cells in culture and in tumor-associated lymphangiogenesis, and podoplanin deficiency results in congenital lymphedema and impaired lymphatic vascular patterning. However, research into the biological importance of podoplanin has been hampered by the lack of a generally available antibody against the human protein, and its expression in normal tissues and in human malignancies has remained unclear. We generated a human podoplanin-Fc fusion protein and found that the commercially available mouse monoclonal antibody D2-40 specifically recognized human podoplanin, as assessed by enzyme-linked immunosorbent assay and Western blot analyses. We found that, in addition to lymphatic endothelium, podoplanin was also expressed by peritoneal mesothelial cells, osteocytes, glandular myoepithelial cells, ependymal cells, and by stromal reticular cells and follicular dendritic cells of lymphoid organs. These findings were confirmed in normal mouse tissues with anti-podoplanin antibody 8.1.1. Podoplanin was also strongly expressed by granulosa cells in normal ovarian follicles, and by ovarian dysgerminomas and granulosa cell tumors. Although podoplanin was primarily absent from normal human epidermis, its expression was strongly induced in 22 of 28 squamous cell carcinomas studied. These findings suggest a potential role of podoplanin in tumor progression, and they also identify the first commercially available antibody for the specific staining of a defined lymphatic marker in archival human tissue sections, thereby enabling more widespread studies of tumor lymphangiogenesis in human cancers.

Fibrillar collagen is the most abundant extracellular matrix (ECM) constituent which maintains the structure of most interstitial tissues and organs, including skin, gut, and breast. Density and spatial alignment of the three-dimensional (3D) collagen architecture define mechanical tissue properties, i.e. stiffness and porosity, which guide or oppose cell migration and positioning in different contexts, such as morphogenesis, regeneration, immune response, and cancer progression. To reproduce interstitial cell movement in vitro with high in vivo fidelity, 3D collagen lattices are being reconstituted from extracted collagen monomers, resulting in the reassembly of a fibrillar meshwork of defined porosity and stiffness. With a focus on tumor invasion studies, we here evaluate different in vitro collagen-based cell invasion models, employing either pepsinized or un-pepsinized collagen extracts, and compare their structure to connective tissue in vivo, including mouse dermis and mammary gland, chick chorioallantoic membrane (CAM), and human dermis. Using confocal reflection and two-photon-excited second harmonic generation (SHG) microscopy, we here show that, depending on the collagen source, in vitro models yield homogeneous fibrillar texture with variable pore sizes, whereas all in vivo scaffolds comprise a range from low- to high-density fibrillar networks and heterogeneous pore sizes within the same tissue. Future in-depth comparison of structure and physical properties between 3D ECM-based models in vitro and in vivo are mandatory to better understand the mechanisms and limits of interstitial cell movements in distinct tissue environments.

Early during development, one of the first indications that lymphangiogenesis has begun is the polarized expression of the homeobox gene Prox1 in a subpopulation of venous endothelial cells. It has been shown previously that Prox1 expression in the cardinal vein promotes and maintains the budding of endothelial cells that will form the lymphatic vascular system. Prox1-deficient mice are devoid of lymphatic vasculature, and in these animals endothelial cells fail to acquire the lymphatic phenotype; instead, they remain as blood vascular endothelium. To investigate whether Prox1 is sufficient to induce a lymphatic fate in blood vascular endothelium, Prox1 cDNA was ectopically expressed by adenoviral gene transfer in primary human blood vascular endothelial cells and by transient plasmid cDNA transfection in immortalized microvascular endothelial cells. Transcriptional profiling combined with quantitative real-time reverse transcriptionpolymerase chain reaction and Western blotting analyses revealed that Prox1 expression up-regulated the lymphatic endothelial cell markers podoplanin and vascular endothelial growth factor receptor-3. Conversely, genes such as laminin, vascular endothelial growth factor-C, neuropilin-1, and intercellular adhesion molecule-1, whose expression has been associated with the blood vascular endothelial cell phenotype, were down-regulated. These results were confirmed by the use of specific antibodies against some of these markers in sections of embryonic and adult tissues. These findings validate our previous proposal that Prox1 is a key player in the molecular pathway leading to the formation of lymphatic vasculature and identify Prox1 as a master switch in the program specifying lymphatic endothelial cell fate. That a single gene product was sufficient to re-program the blood vascular endothelium toward a lymphatic phenotype corroborates the close relationship between these two vascular systems and also suggests that during evolution, the lymphatic vasculature originated from the blood vasculature by the additional expression of only a few gene products such as Prox1.

In mammals, the lymphatic vascular system develops by budding of lymphatic progenitor endothelial cells from embryonic veins to form a distinct network of draining vessels with important functions in the immune response and in cancer metastasis. However, the lineage-specific molecular characteristics of blood vascular versus lymphatic endothelium have remained poorly defined. We isolated lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BVECs) by immunomagnetic isolation directly from human skin. Cultured LECs but not BVECs expressed the lymphatic markers Prox1 and LYVE-1 and formed LYVE-1-positive vascular tubes after implantation in vivo. Transcriptional profiling studies revealed increased expression of several extracellular matrix and adhesion molecules in BVECs, including versican, collagens, laminin, and N-cadherin, and of the growth factor receptors endoglin and vascular endothelial growth factor receptor-1/Flt-1. Differential immunostains of human skin confirmed the blood vessel-specific expression of these genes. During embryonic development, endoglin expression was gradually down-regulated on lymphatic endothelium whereas vascular endothelial growth factor receptor-1 was absent from lymphatics. We also identified several genes with specific expression in LECs. These results demonstrate that some lineage-specific genes are only expressed during distinct developmental stages and they identify new molecular markers for blood vascular and lymphatic endothelium with im- The lymphatic system consists of a vascular network of thin-walled capillaries that drain protein-rich lymph from the extracellular spaces within most organs and that play major roles in the immune response and in tumor metastasis.1,2 Lymphatic vessels provide the conduit for antigen-presenting cells from the organ exposed to antigens to the regional lymph nodes, involving active recruitment of antigen-presenting cells by chemokines and other mediators secreted by lymphatic endothelium.3 Moreover, the early dissemination of malignant tumors frequently occurs via lymphatic vessels to regional lymph nodes, and the recent discovery of active tumor lymphangiogenesis and its role in cancer metastasis has drawn considerable attention to the molecular mechanisms that control activation and proliferation of lymphatic endothelium. 2,4

Vascular endothelial growth factor-A (VEGF-A) expression is up-regulated in several inflammatory diseases including psoriasis, delayed-type hypersensitivity (DTH) reactions, and rheumatoid arthritis. To directly characterize the biologic function of VEGF-A in inflammation, we evaluated experimental DTH reactions induced in the ear skin of transgenic mice that overexpress VEGF-A specifically in the epidermis. VEGF-A transgenic mice underwent a significantly increased inflammatory response that persisted for more than 1 month, whereas inflammation returned to baseline levels within 7 days in wild-type mice. Inflammatory lesions in VEGF-A transgenic mice closely resembled human psoriasis and were characterized by epidermal hyperplasia, impaired epidermal differentiation, and accumulation of dermal CD4 ؉ T-lymphocytes and epidermal CD8 ؉ lymphocytes. Surprisingly, VEGF-A also promoted lymphatic vessel proliferation and enlargement, which might contribute to the increased inflammatory response, as lymphatic vessel enlargement was also detected in human psoriatic skin lesions. Combined systemic treatment with blocking antibodies against VEGF receptor-1 (VEGFR-1) and VEGFR-2 potently inhibited inflammation and also decreased lymphatic vessel size. Together, these findings reveal a central role of VEGF-A in promoting lymphatic enlargement, vascular hyperpermeability, and leukocyte recruitment, thereby leading to persistent chronic inflammation. They also indicate that inhibition of VEGF-A bioactivity might be a new approach to anti-inflammatory therapy. IntroductionVascular endothelial growth factor-A (VEGF-A) is a homodimeric heparin-binding glycoprotein that occurs in at least 4 isoforms of 121, 165, 189, and 201 amino acids, as a result of alternative splicing (corresponding murine proteins are one amino acid shorter). VEGF-A binds to the 2 type III receptor tyrosine kinases VEGF receptor-1 (VEGFR-1, Flt-1) and VEGFR-2 (KDR or Flk-1), which are primarily expressed by vascular endothelial cells. 1,2 VEGF-A165 also binds to the nonkinase receptors neuropilin 1 and 2. 3 VEGF-A stimulates endothelial cell migration, proliferation, and survival in vitro and promotes microvascular permeability and angiogenesis in vivo. 4,5 Previous studies have revealed that VEGF-A expression by epidermal keratinocytes and endothelial expression of VEGF receptors are up-regulated in several inflammatory conditions including psoriasis, 6 delayed-type hypersensitivity reactions, 7 and bullous diseases. 8 However, the biologic importance of VEGF in the pathogenesis of chronic inflammation has remained unclear.The lymphatic vascular system is composed of a network of thin-walled capillaries that drain protein-rich lymph from the extracellular space and thereby maintain normal tissue pressure. 9 Moreover, lymphatic vessels play an important role in the afferent phase of the immune response. 10 Little is known about the role of the lymphatic vascular system in the control of chronic inflammation, however, due to a lack of reliable markers and t...

The present, revised guidelines on lipedema were developed under the auspices of and funded by the German Society of Phlebology (DGP). The recommendations are based on a systematic literature search and the consensus of eight medical societies and working groups. The guidelines contain recommendations with respect to diagnosis and management of lipedema. The diagnosis is established on the basis of medical history and clinical findings. Characteristically, there is a localized, symmetrical increase in subcutaneous adipose tissue in arms and legs that is in marked disproportion to the trunk. Other findings include edema, easy bruising, and increased tenderness. Further diagnostic tests are usually reserved for special cases that require additional workup. Lipedema is a chronic, progressive disorder marked by the individual variability and unpredictability of its clinical course. Treatment consists of four therapeutic mainstays that should be combined as necessary and address current clinical symptoms: complex physical therapy (manual lymphatic drainage, compression therapy, exercise therapy, and skin care), liposuction and plastic surgery, diet, and physical activity, as well as psychotherapy if necessary. Surgical procedures are indicated if - despite thorough conservative treatment - symptoms persist, or if there is progression of clinical findings and/or symptoms. If present, morbid obesity should be therapeutically addressed prior to liposuction.

Using the presented method the surface pO(2) distribution can be mapped with a high temporal resolution of approximately 100 ms and a spatial resolution of at least 25 mu m. Moreover, the transparent sensor allows the simultaneous visualization of the underlying microvasculature.