Local induction of lymphangiogenesis with engineered fibrin-binding VEGF-C promotes wound healing by increasing immune cell trafficking and matrix remodeling

Lymphangiogenesis occurs in inflammation and wound healing, yet its functional roles in these processes are not fully understood. Consequently, clinically relevant strategies for therapeutic lymphangiogenesis remain underdeveloped, particularly using growth factors. To achieve controlled, local capillary lymphangiogenesis with protein engineering and determine its effects on fluid clearance, leukocyte trafficking, and wound healing, we developed a fibrin-binding variant of vascular endothelial growth factor C (FB-VEGF-C) that is slowly released upon demand from infiltrating cells. Using a novel wound healing model, we show that implanted fibrin containing FB-VEGF-C, but not free VEGF-C, could stimulate local lymphangiogenesis in a dose-dependent manner. Importantly, the effects of FB-VEGF-C were restricted to lymphatic capillaries, with no apparent changes to blood vessels and downstream collecting vessels. Leukocyte intravasation and trafficking to lymph nodes were increased in hyperplastic lymphatics, while fluid clearance was maintained at physiological levels. In diabetic wounds, FB-VEGF-C-induced lymphangiogenesis increased extracellular matrix deposition and granulation tissue thickening, indicators of improved wound healing. Together, these results indicate that FB-VEGF-C is a promising strategy for inducing lymphangiogenesis locally, and that such lymphangiogenesis can promote wound healing by enhancing leukocyte trafficking without affecting downstream lymphatic collecting vessels. (C) 2017 The Authors. Published by Elsevier Ltd

Malaria risk and access to prevention and treatment in the paddies of the Kilombero Valley, Tanzania

Background: The Kilombero Valley is a highly malaria-endemic agricultural area in south-eastern Tanzania. Seasonal flooding of the valley is favourable to malaria transmission. During the farming season, many households move to distant field sites (shamba in Swahili) in the fertile river floodplain for the cultivation of rice. In the shamba, people live for several months in temporary shelters, far from the nearest health services. This study assessed the impact of seasonal movements to remote fields on malaria risk and treatment-seeking behaviour. Methods: A longitudinal study followed approximately 100 randomly selected farming households over six months. Every household was visited monthly and whereabouts of household members, activities in the fields, fever cases and treatment seeking for recent fever episodes were recorded. Results: Fever incidence rates were lower in the shamba compared to the villages and moving to the shamba did not increase the risk of having a fever episode. Children aged 1-4 years, who usually spend a considerable amount of time in the shamba with their caretakers, were more likely to have a fever than adults (odds ratio = 4.47, 95 confidence interval 2.35-8.51). Protection with mosquito nets in the fields was extremely good (98 antimalarials was uncommon. Despite the long distances to health services, 55.8 health facility, while home-management was less common (37 17.4-50.5). Conclusion: Living in the shamba does not appear to result in a higher fever-risk. Mosquito nets usage and treatment of fever in health facilities reflect awareness of malaria. Inability to obtain drugs in the fields may contribute to less irrational use of drugs but may pose an additional burden on poor farming households. A comprehensive approach is needed to improve access to treatment while at the same time assuring rational use of medicines and protecting fragile livelihoods

HIF-driven SF3B1 induces KHK-C to enforce fructolysis and heart disease.

Fructose is a major component of dietary sugar and its overconsumption exacerbates key pathological features of metabolic syndrome. The central fructose-metabolising enzyme is ketohexokinase (KHK), which exists in two isoforms: KHK-A and KHK-C, generated through mutually exclusive alternative splicing of KHK pre-mRNAs. KHK-C displays superior affinity for fructose compared with KHK-A and is produced primarily in the liver, thus restricting fructose metabolism almost exclusively to this organ. Here we show that myocardial hypoxia actuates fructose metabolism in human and mouse models of pathological cardiac hypertrophy through hypoxia-inducible factor 1α (HIF1α) activation of SF3B1 and SF3B1-mediated splice switching of KHK-A to KHK-C. Heart-specific depletion of SF3B1 or genetic ablation of Khk, but not Khk-A alone, in mice, suppresses pathological stress-induced fructose metabolism, growth and contractile dysfunction, thus defining signalling components and molecular underpinnings of a fructose metabolism regulatory system crucial for pathological growth

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy

Lymphatic vessels regulate immune microenvironments in human and murine melanoma

Lymphatic remodeling in tumor microenvironments correlates with progression and metastasis, and local lymphatic vessels play complex and poorly understood roles in tumor immunity. Tumor lymphangiogenesis is associated with increased immune suppression, yet lymphatic vessels are required for fluid drainage and immune cell trafficking to lymph nodes, where adaptive immune responses are mounted. Here, we examined the contribution of lymphatic drainage to tumor inflammation and immunity using a mouse model that lacks dermal lymphatic vessels (K14-VEGFR3-Ig mice). Melanomas implanted in these mice grew robustly, but exhibited drastically reduced cytokine expression and leukocyte infiltration compared with those implanted in control animals. In the absence of local immune suppression, transferred cytotoxic T cells more effectively controlled tumors in K14-VEGFR3-Ig mice than in control mice. Furthermore, gene expression analysis of human melanoma samples revealed that patient immune parameters are markedly stratified by levels of lymphatic markers. This work suggests that the establishment of tumor-associated inflammation and immunity critically depends on lymphatic vessel remodeling and drainage. Moreover, these results have implications for immunotherapies, the efficacies of which are regulated by the tumor immune microenvironment

A Risk-benefit Analysis of Prophylactic Anticoagulation for Patients with Metastatic Germ Cell Tumours Undergoing First-line Chemotherapy

BACKGROUND It remains unclear which patients with metastatic germ cell tumours (mGCTs) need prophylactic anticoagulation to prevent venous thromboembolic events (VTEs). OBJECTIVE To assess the risk and onset of VTEs stratified by risk factors. DESIGN, SETTING, AND PARTICIPANTS This multi-institutional retrospective dataset included mGCT patients treated with first-line platinum-based chemotherapy. INTERVENTION Patients with prophylactic anticoagulation were excluded. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS A regression analysis was performed to select risk factors for VTEs. The simulated number needed to treat (NNT) and the number needed to harm (NNH) with prophylactic anticoagulation were calculated based on the cumulative incidences retrieved from this study and hazard rates of recently published trials describing the efficacy of prophylactic anticoagulation to prevent VTEs and the risk of bleeding events. RESULTS AND LIMITATIONS From 1120 patients, 121 (11%) had a VTE, which occurred prior to chemotherapy in 49 (4%) and on or after chemotherapy in 72 (6%). Six patients (<1%) had a bleeding event without anticoagulation. After backward regression, the one risk factor for a VTE during or after chemotherapy was the use of a venous access device. The simulated cumulative VTE incidence from prophylactic anticoagulation for patients on or after chemotherapy would translate into an NNT of 45 (95% confidence interval [CI] 36-56) and an NNH of 186 (95% CI 87-506). Limitations are mainly related to the retrospective nature of the study. CONCLUSIONS The mGCTs associated VTEs are most common before and during, but not after, chemotherapy. Avoiding venous access device and/or prophylactic anticoagulation with an acceptable risk-benefit profile may decrease VTE occurring on chemotherapy. PATIENT SUMMARY We found that venous thromboembolic events (VTEs) occur rarely after chemotherapy. Based on experience of prophylactic anticoagulation in other cancers, we conclude that the risk of VTE in men undergoing chemotherapy for metastatic germ cell tumours can be decreased by thromboprophylaxis with a reasonable risk-benefit profile and by avoidance of venous access devices

A molecular framework for light and gibberellin control of cell elongation

Cell elongation during seedling development is antagonistically regulated by light and gibberellins (GAs). Light induces photomorphogenesis, leading to inhibition of hypocotyl growth, whereas GAs promote etiolated growth, characterized by increased hypocotyl elongation. The mechanism underlying this antagonistic interaction remains unclear. Here we report on the central role of the Arabidopsis thaliana nuclear transcription factor PIF4 (encoded by PHYTOCHROME INTERACTING FACTOR 4) in the positive control of genes mediating cell elongation and show that this factor is negatively regulated by the light photoreceptor phyB (ref. 4) and by DELLA proteins that have a key repressor function in GA signalling. Our results demonstrate that PIF4 is destabilized by phyB in the light and that DELLAs block PIF4 transcriptional activity by binding the DNA-recognition domain of this factor. We show that GAs abrogate such repression by promoting DELLA destabilization, and therefore cause a concomitant accumulation of free PIF4 in the nucleus. Consistent with this model, intermediate hypocotyl lengths were observed in transgenic plants over-accumulating both DELLAs and PIF4. Destabilization of this factor by phyB, together with its inactivation by DELLAs, constitutes a protein interaction framework that explains how plants integrate both light and GA signals to optimize growth and development in response to changing environments