Postnatal Deletion of Podoplanin in Lymphatic Endothelium Results in Blood Filling of the Lymphatic System and Impairs Dendritic Cell Migration to Lymph Nodes.

OBJECTIVE: The lymphatic vascular system exerts major physiological functions in the transport of interstitial fluid from peripheral tissues back to the blood circulation and in the trafficking of immune cells to lymph nodes. Previous studies in global constitutive knockout mice for the lymphatic transmembrane molecule podoplanin reported perinatal lethality and a complex phenotype with lung abnormalities, cardiac defects, lymphedema, blood-filled lymphatic vessels, and lack of lymph node organization, reflecting the importance of podoplanin expression not only by the lymphatic endothelium but also by a variety of nonendothelial cell types. Therefore, we aimed to dissect the specific role of podoplanin expressed by adult lymphatic vessels. APPROACH AND RESULTS: We generated an inducible, lymphatic-specific podoplanin knockout mouse model (PdpnΔLEC) and induced gene deletion postnatally. PdpnΔLEC mice were viable, and their lymphatic vessels appeared morphologically normal with unaltered fluid drainage function. Intriguingly, PdpnΔLEC mice had blood-filled lymph nodes and vessels, most frequently in the neck and axillary region, and displayed a blood-filled thoracic duct, suggestive of retrograde filling of blood from the blood circulation into the lymphatic system. Histological and fluorescence-activated cell sorter analyses revealed normal lymph node organization with the presence of erythrocytes within lymph node lymphatic vessels but not surrounding high endothelial venules. Moreover, fluorescein isothiocyanate painting experiments revealed reduced dendritic cell migration to lymph nodes in PdpnΔLEC mice. CONCLUSIONS: These results reveal an important role of podoplanin expressed by lymphatic vessels in preventing postnatal blood filling of the lymphatic vascular system and in contributing to efficient dendritic cell migration to the lymph nodes.

Keratinocyte-Expressed Podoplanin is Dispensable for Multi-Step Skin Carcinogenesis.

Podoplanin is a small transmembrane mucin-like glycoprotein that plays a crucial role in the development of the lung, heart and lymphatic vascular system. Its expression is upregulated in several types of human carcinomas and podoplanin levels in squamous cell carcinomas (SCCs) of the oral cavity and the lung correlate with cancer invasiveness, lymph node metastasis and shorter survival time of patients, indicating that podoplanin promotes tumor progression. However, its role during the early stages of carcinogenesis remain unclear. We generated mice with a specific deletion of podoplanin in epidermal keratinocytes (K5-Cre;Pdpnflox/flox mice) and subjected them to a multistep chemical skin carcinogenesis regimen. The rate of tumor initiation; the number, size and differentiation of tumors; and the malignant transformation rate were comparable in K5-Cre;Pdpnflox/flox mice and Pdpnflox/flox control mice. However, tumor cell invasion was reduced in K5-Cre;Pdpnflox/flox mice, in particular single cell invasion. Quantitative immunofluorescence analyses revealed that peritumoral lymphangiogenesis was reduced in K5-Cre;Pdpnflox/flox mice, whereas there were no major changes of tumor-associated immune cell subpopulations. Thus, keratinocyte-expressed podoplanin is dispensable for the early steps of skin carcinogenesis but contributes to the progression of established tumors.

An important role of podoplanin in hair follicle growth.

Podoplanin (PDPN) is a glycoprotein that is expressed by various cell types, including keratinocytes, fibroblasts, and lymphatic endothelial cells. We found that PDPN is expressed in the hair follicle (HF) keratinocyte region and HF stem cell area during the late anagen phase but not during the telogen phase in mice. Importantly, keratinocyte-specific PDPN deletion in mice (K5-Cre;PDPNflox/flox) promoted anagen HF growth after depilation-induced HF regeneration as compared to control mice. RNA sequencing, followed by gene ontology analysis, showed down-regulation of focal adhesion and extracellular matrix interaction pathways in HF stem cells isolated from K5-Cre;PDPNflox/flox mice as compared to control mice. Furthermore, HF keratinocytes isolated from K5-Cre;PDPNflox/flox mice exhibited a decreased ability to interact with collagen type I in cell adhesion assays. Taken together, these results show that PDPN deletion promotes HF cycling, possibly via reduced focal adhesion and concomitantly enhanced migration of HF stem cells towards the bulb region. They also indicate potential new therapeutic strategies for the treatment of conditions associated with hair loss.

Antibody-Mediated Delivery of VEGFC Ameliorates Experimental Chronic Colitis.

Crohn's disease (CD) and ulcerative colitis (UC) are two distinct forms of inflammatory bowel disease (IBD) characterized by an expanded lymphatic network with impaired functionality both in mouse models and in human patients. In this study, we investigated whether targeted delivery of the pro-lymphangiogenic vascular endothelial growth factor C (VEGFC) to the site of inflammation may represent a new, clinically feasible strategy for treating IBD. To achieve targeting of inflamed tissue, we developed a fusion protein consisting of human VEGFC fused to the F8 antibody (F8-VEGFC), which specifically binds to the extradomain A (EDA) of fibronectin, a spliced isoform almost exclusively expressed in inflamed tissues. The therapeutic activity of intravenously administered F8-VEGFC, compared to a targeted construct lacking VEGFC (F8-SIP), was investigated in a mouse model of dextran sodium sulfate (DSS)-induced colitis. The presence of EDA fibronectin was detected in both human and mouse inflamed colon tissue. Biodistribution studies of radiolabeled F8-VEGFC revealed a specific accumulation of the antibody in the colon of DSS-administered mice, as compared to an untargeted VEGFC fusion protein (KSF-VEGFC) (binding the irrelevant hen egg lysozyme antigen). Systemic treatment with F8-VEGFC significantly reduced the clinical and histological signs of inflammation, expanded the lymphatic vascular network, reduced the density of immune cells, and also decreased the expression of inflammatory cytokines in the inflamed colon. Overall, these results reveal that administration of F8-VEGFC represents a novel and promising approach for the treatment of IBD.