Lymphatic Capillaries in Aging.

The lymphatic system is responsible for fluid drainage from almost every organ in the body. It sustains tissue homeostasis and is also a central part of the immune system. With the discovery of cell-specific markers and transgenic mouse models, it has become possible to gain some insight into the developmental and functional roles of lymphatic endothelial cells (LECs). Only recently, a more direct regulatory role has been assigned to LECs in their functions in immunity responses and chronic diseases. Here, we discuss the changes occurring in aged lymphatic system and the role of lymphatic capillaries in some age-related diseases and experimental animal models.

LIM and SH3 protein 1 (LASP-1): A novel link between the slit membrane and actin cytoskeleton dynamics in podocytes.

The foot processes of podocytes exhibit a dynamic actin cytoskeleton, which maintains their complex cell structure and antagonizes the elastic forces of the glomerular capillary. Interdigitating secondary foot processes form a highly selective filter for proteins in the kidney, the slit membrane. Knockdown of slit membrane components such as Nephrin or Neph1 and cytoskeletal adaptor proteins such as CD2AP in mice leads to breakdown of the filtration barrier with foot process effacement, proteinuria, and early death of the mice. Less is known about the crosstalk between the slit membrane-associated proteins and cytoskeletal components inside the podocyte foot processes. Our study shows that LASP-1, an actin-binding protein, is highly expressed in podocytes. Electron microscopy studies demonstrate that LASP-1 is found at the slit membrane suggesting a role in anchoring slit membrane components to the actin cytoskeleton. Live cell imaging experiments with transfected podocytes reveal that LASP-1 is either part of a highly dynamic granular complex or a static, actin cytoskeleton-bound protein. We identify CD2AP as a novel LASP-1 binding partner that regulates its association with the actin cytoskeleton. Activation of the renin-angiotensin-aldosterone system, which is crucial for podocyte function, leads to phosphorylation and altered localization of LASP-1. In vivo studies using the Drosophila nephrocyte model indicate that Lasp is necessary for the slit membrane integrity and functional filtration.

Lymphangiogenesis in a mouse model of renal transplant rejection extends life span of the recipients.

Renal allograft rejection can be prevented by immunological tolerance, which may be associated with de novo formed lymphatic vessels in the donor kidney after transplantation in man. A suitable mouse model of renal allograft rejection in which lymphangiogenesis can be deliberately induced in the graft is critical for elucidating the mechanisms responsible for the association between attenuated transplant rejection and abundance of lymphatic vessels. Here we describe the development of a novel mouse model of rapid renal transplant rejection in which transgenic induction of lymphangiogenesis in the immune-incompatible graft greatly extends its survival time. Thus, our novel approach may facilitate exploitation of lymphangiogenesis in the grafted organ.

Generation of blood vessel organoids from human pluripotent stem cells.

Blood vessels are fundamental to animal life and have critical roles in many diseases, such as stroke, myocardial infarction and diabetes. The vasculature is formed by endothelial cells that line the vessel and are covered with mural cells, specifically pericytes in smaller vessels and vascular smooth muscle cells (vSMCs) in larger-diameter vessels. Both endothelial cells and mural cells are essential for proper blood vessel function and can be derived from human pluripotent stem cells (hPSCs). Here, we describe a protocol to generate self-organizing 3D human blood vessel organoids from hPSCs that exhibit morphological, functional and molecular features of human microvasculature. These organoids are differentiated via mesoderm induction of hPSC aggregates and subsequent differentiation into endothelial networks and pericytes in a 3D collagen I-Matrigel matrix. Blood vessels form within 2-3 weeks and can be further grown in scalable suspension culture. Importantly, in vitro-differentiated human blood vessel organoids transplanted into immunocompromised mice gain access to the mouse circulation and specify into functional arteries, arterioles and veins.

Human blood vessel organoids as a model of diabetic vasculopathy.

The increasing prevalence of diabetes has resulted in a global epidemic1. Diabetes is a major cause of blindness, kidney failure, heart attacks, stroke and amputation of lower limbs. These are often caused by changes in blood vessels, such as the expansion of the basement membrane and a loss of vascular cells2-4. Diabetes also impairs the functions of endothelial cells5 and disturbs the communication between endothelial cells and pericytes6. How dysfunction of endothelial cells and/or pericytes leads to diabetic vasculopathy remains largely unknown. Here we report the development of self-organizing three-dimensional human blood vessel organoids from pluripotent stem cells. These human blood vessel organoids contain endothelial cells and pericytes that self-assemble into capillary networks that are enveloped by a basement membrane. Human blood vessel organoids transplanted into mice form a stable, perfused vascular tree, including arteries, arterioles and venules. Exposure of blood vessel organoids to hyperglycaemia and inflammatory cytokines in vitro induces thickening of the vascular basement membrane. Human blood vessels, exposed in vivo to a diabetic milieu in mice, also mimic the microvascular changes found in patients with diabetes. DLL4 and NOTCH3 were identified as key drivers of diabetic vasculopathy in human blood vessels. Therefore, organoids derived from human stem cells faithfully recapitulate the structure and function of human blood vessels and are amenable systems for modelling and identifying the regulators of diabetic vasculopathy, a disease that affects hundreds of millions of patients worldwide.

ARP3 Controls the Podocyte Architecture at the Kidney Filtration Barrier.

Podocytes, highly specialized epithelial cells, build the outer part of the kidney filtration barrier and withstand high mechanical forces through a complex network of cellular protrusions. Here, we show that Arp2/3-dependent actin polymerization controls actomyosin contractility and focal adhesion maturation of podocyte protrusions and thereby regulates formation, maintenance, and capacity to adapt to mechanical requirements of the filtration barrier. We find that N-WASP-Arp2/3 define the development of complex arborized podocyte protrusions in vitro and in vivo. Loss of dendritic actin networks results in a pronounced activation of the actomyosin cytoskeleton and the generation of over-maturated but less efficient adhesion, leading to detachment of podocytes. Our data provide a model to explain podocyte protrusion morphology and their mechanical stability based on a tripartite relationship between actin polymerization, contractility, and adhesion.

Expression of 15-lipoxygenase-1 in Merkel cell carcinoma is linked to advanced disease.

BACKGROUND: The purpose of this study was to determine whether the expression of 15-lipoxygenase-1 (ALOX15) in primary tumour specimens predicts lymph node metastasis and subsequently clinical outcome in Merkel cell carcinoma (MCC) patients. METHODS: A retrospective medical chart review of 33 patients was performed between 1994 and 2014. Eleven out of 33 (33%) Patients with primary MCC stages I and II were categorised as group I. Twenty two out of 33 (67%) Patients with regional lymph node metastases and/or distant metastases were defined as group II. All available tumour samples were immunostained for ALOX15, Podoplanin and MCPyV large T-protein antibody. RESULTS: ALOX15 expression was observed in 19/23 (83%) primary tumour samples and in all lymph node metastasis. Primary tumours in patients with stage III and IV disease showed a higher expression rate of ALOX15 compared to patients with early stage disease (11/12 (92%) and 8/11 (73%), respectively). In group I, five patients (45%) were MCPyV positive, whereas in group II, 15 patients (68%) were MCPyV positive. The median lymphatic vessel density in ALOX15 negative group I primary tumour samples was lower compared to the median lymphatic vessel density in ALOX15 positive group I primary tumour probes (2.7 range, 1-4.3 vs 4.7 range, 4.0-7.3). Furthermore, all 17 samples of MCC metastases showed ALOX15 expression with a median lymphatic vessel density (not lymph node metastases) of 5.3 (range 2.0-7.3). CONCLUSION: In the current study, we were able to show ALOX15 expression in the primary MCC sample and the metastasis sample. Based on the findings of the current study, expression rate of ALOX15 in primary MCC and metastases is possibly linked to an increased lymphatic vessel density.

Breast cancer metastasis through the lympho-vascular system.

Breast cancer metastasizes through the lymphovascular system to the regional lymph nodes in the axilla and to both visceral and non-visceral sites. Renewed interest in the route by which tumor cells gain access to blood and lymphatic capillaries are the subject of research at mechanical, anatomic, pathologic, genetic, epidemiologic and molecular levels. Two papers presented at the 7th International Symposium on Cancer Metastasis in San Francisco showed tumor cells entering the systemic circulation through the sentinel lymph node. This information challenges the current paradigm where clinicians believe that access is gained through intra- and peri-tumoral blood vessels and that metastasis to axillary lymph nodes is an interesting epi-phenomenon. The sentinel lymph node era has changed the modern surgical approach to the axilla and the basis of this change is summarized in this paper. A new approach to the management of axillary metastases after systemic therapy relies on determining whether there is a complete pathologic response; if no tumor is found in the previously biopsied node, a complete axillary lymph node dissection may be avoided. African American women seem to inherit a trait from West African ancestors and tend to develop more lethal types of breast cancer. These tumors may have a molecular machinery that enhances their ability to metastasize to visceral sites and future research may unearth the mechanisms for this phenomenon.

Lymphatic exosomes promote dendritic cell migration along guidance cues.

Lymphatic endothelial cells (LECs) release extracellular chemokines to guide the migration of dendritic cells. In this study, we report that LECs also release basolateral exosome-rich endothelial vesicles (EEVs) that are secreted in greater numbers in the presence of inflammatory cytokines and accumulate in the perivascular stroma of small lymphatic vessels in human chronic inflammatory diseases. Proteomic analyses of EEV fractions identified >1,700 cargo proteins and revealed a dominant motility-promoting protein signature. In vitro and ex vivo EEV fractions augmented cellular protrusion formation in a CX3CL1/fractalkine-dependent fashion and enhanced the directional migratory response of human dendritic cells along guidance cues. We conclude that perilymphatic LEC exosomes enhance exploratory behavior and thus promote directional migration of CX3CR1-expressing cells in complex tissue environments.

Residual urinary extracellular vesicles in ultracentrifugation supernatants after hydrostatic filtration dialysis enrichment.

Urinary extracellular vesicles (UEVs) appear an ideal source of biomarkers for kidney and urogenital diseases. The majority of protocols designed for their isolation are based on differential centrifugation steps. However, little is still known of the type and amount of vesicles left in the supernatant. Here we used an isolation protocol for UEVs which uses hydrostatic filtration dialysis as first pre-enrichment step, followed by differential centrifugation. Transmission electron microscopy (TEM), mass spectrometry (MS), western blot, ELISA assays and tuneable resistive pulse sensing (TRPS) were used to characterise and quantify UEVs in the ultracentrifugation supernatant. TEM showed the presence of a variety of small size vesicles in the supernatant while protein identification by MS matched accurately with the protein list available in Vesiclepedia. Screening and relative quantification for specific vesicle markers showed that the supernatant was preferentially positive for CD9 and TSG101. ELISA tests for quantification of exosome revealed that 14%, was left in the supernatant with a particle diameter of 110 nm and concentration of 1.54 × 1010/ml. Here we show a comprehensive characterisation of exosomes and other small size urinary vesicles which the conventional differential centrifugation protocol may lose.

Heme drives hemolysis-induced susceptibility to infection via disruption of phagocyte functions.

Hemolysis drives susceptibility to bacterial infections and predicts poor outcome from sepsis. These detrimental effects are commonly considered to be a consequence of heme-iron serving as a nutrient for bacteria. We employed a Gram-negative sepsis model and found that elevated heme levels impaired the control of bacterial proliferation independently of heme-iron acquisition by pathogens. Heme strongly inhibited phagocytosis and the migration of human and mouse phagocytes by disrupting actin cytoskeletal dynamics via activation of the GTP-binding Rho family protein Cdc42 by the guanine nucleotide exchange factor DOCK8. A chemical screening approach revealed that quinine effectively prevented heme effects on the cytoskeleton, restored phagocytosis and improved survival in sepsis. These mechanistic insights provide potential therapeutic targets for patients with sepsis or hemolytic disorders.

Diversified actin protrusions promote environmental exploration but are dispensable for locomotion of leukocytes.

Most migrating cells extrude their front by the force of actin polymerization. Polymerization requires an initial nucleation step, which is mediated by factors establishing either parallel filaments in the case of filopodia or branched filaments that form the branched lamellipodial network. Branches are considered essential for regular cell motility and are initiated by the Arp2/3 complex, which in turn is activated by nucleation-promoting factors of the WASP and WAVE families. Here we employed rapid amoeboid crawling leukocytes and found that deletion of the WAVE complex eliminated actin branching and thus lamellipodia formation. The cells were left with parallel filaments at the leading edge, which translated, depending on the differentiation status of the cell, into a unipolar pointed cell shape or cells with multiple filopodia. Remarkably, unipolar cells migrated with increased speed and enormous directional persistence, while they were unable to turn towards chemotactic gradients. Cells with multiple filopodia retained chemotactic activity but their migration was progressively impaired with increasing geometrical complexity of the extracellular environment. These findings establish that diversified leading edge protrusions serve as explorative structures while they slow down actual locomotion.

The brain-tumor related protein podoplanin regulates synaptic plasticity and hippocampus-dependent learning and memory.

INTRODUCTION: Podoplanin is a cell-surface glycoprotein constitutively expressed in the brain and implicated in human brain tumorigenesis. The intrinsic function of podoplanin in brain neurons remains however uncharacterized. MATERIALS AND METHODS: Using an established podoplanin-knockout mouse model and electrophysiological, biochemical, and behavioral approaches, we investigated the brain neuronal role of podoplanin. RESULTS: Ex-vivo electrophysiology showed that podoplanin deletion impairs dentate gyrus synaptic strengthening. In vivo, podoplanin deletion selectively impaired hippocampus-dependent spatial learning and memory without affecting amygdala-dependent cued fear conditioning. In vitro, neuronal overexpression of podoplanin promoted synaptic activity and neuritic outgrowth whereas podoplanin-deficient neurons exhibited stunted outgrowth and lower levels of p-Ezrin, TrkA, and CREB in response to nerve growth factor (NGF). Surface Plasmon Resonance data further indicated a physical interaction between podoplanin and NGF. DISCUSSION: This work proposes podoplanin as a novel component of the neuronal machinery underlying neuritogenesis, synaptic plasticity, and hippocampus-dependent memory functions. The existence of a relevant cross-talk between podoplanin and the NGF/TrkA signaling pathway is also for the first time proposed here, thus providing a novel molecular complex as a target for future multidisciplinary studies of the brain function in the physiology and the pathology. Key messages Podoplanin, a protein linked to the promotion of human brain tumors, is required in vivo for proper hippocampus-dependent learning and memory functions. Deletion of podoplanin selectively impairs activity-dependent synaptic strengthening at the neurogenic dentate-gyrus and hampers neuritogenesis and phospho Ezrin, TrkA and CREB protein levels upon NGF stimulation. Surface plasmon resonance data indicates a physical interaction between podoplanin and NGF. On these grounds, a relevant cross-talk between podoplanin and NGF as well as a role for podoplanin in plasticity-related brain neuronal functions is here proposed.

Blood capillary rarefaction and lymphatic capillary neoangiogenesis are key contributors to renal allograft fibrosis in an ACE inhibition rat model.

Chronic allograft fibrosis is the major cause of graft loss in kidney transplantation. Progression can only be reduced by inhibition of the renin-angiotensin system (RAS). We tested the hypothesis that the protection provided by angiotensin-converting enzyme (ACE) inhibition also decreases capillary rarefaction, lymphangiogenesis, and podocyte injury in allograft fibrosis. Fisher kidneys were transplanted into bilaterally nephrectomized Lewis rats treated with enalapril (60 mg/kg per day) (ACE inhibitor, ACEi) or vehicle. Proteinuria, blood urea nitrogen, and plasma creatinine were regularly assessed, and grafts were harvested for morphological and immunohistological analysis at various times up to 32 wk. In the vehicle group, many new lymphatic capillaries and severe and diffuse mononuclear infiltration of allografts were observed already 1 wk after transplantation. Lymphangiogenesis increased until week 4, by which time inflammatory infiltration became focal. Lymphatic capillaries were often located at sites of inflammation. Progressive interstitial fibrosis, glomerulosclerosis, capillary rarefaction, and proteinuria appeared later, at weeks 4-12 The number of lymphatic capillary cross sections strongly correlated with the interstitial fibrosis score. Podoplanin immunostaining, a marker of healthy podocytes, disappeared from inflamed or sclerotic glomerular areas. ACEi protected from lymphangiogenesis and associated inflammation, preserved glomerular podoplanin protein expression, and reduced glomerulosclerosis, proteinuria, tubulointerstitial fibrosis, and blood capillary rarefaction at 32 wk. In conclusion, ACEi considerably decreased and/or delayed both glomerulosclerosis and tubulointerstitial injury. Prevention of glomerular podoplanin loss and proteinuria could be attributed to the known intraglomerular pressure-lowering effects of ACEi. Reduction of lymphangiogenesis could contribute to amelioration of tubulointerstitial fibrosis and inflammatory infiltration after ACEi.

A flexible, multilayered protein scaffold maintains the slit in between glomerular podocytes.

Vertebrate life critically depends on renal filtration and excretion of low molecular weight waste products. This process is controlled by a specialized cell-cell contact between podocyte foot processes: the slit diaphragm (SD). Using a comprehensive set of targeted KO mice of key SD molecules, we provided genetic, functional, and high-resolution ultrastructural data highlighting a concept of a flexible, dynamic, and multilayered architecture of the SD. Our data indicate that the mammalian SD is composed of NEPHRIN and NEPH1 molecules, while NEPH2 and NEPH3 do not participate in podocyte intercellular junction formation. Unexpectedly, homo- and heteromeric NEPHRIN/NEPH1 complexes are rarely observed. Instead, single NEPH1 molecules appear to form the lower part of the junction close to the glomerular basement membrane with a width of 23 nm, while single NEPHRIN molecules form an adjacent junction more apically with a width of 45 nm. In both cases, the molecules are quasiperiodically spaced 7 nm apart. These structural findings, in combination with the flexibility inherent to the repetitive Ig folds of NEPHRIN and NEPH1, indicate that the SD likely represents a highly dynamic cell-cell contact that forms an adjustable, nonclogging barrier within the renal filtration apparatus.

2015 Homer W. Smith Award: The Podocyte from Periphery to Center Stage.

This overview summarizes selected major developments over the last 40 years in understanding podocyte biology and its involvement in glomerular disease subjectively from my perspective. Serendipity has played a major role in my contributions to investigative nephrology that range from basic mechanisms of immune deposit formation in experimental membranous nephropathy to the role of a microRNA in FSGS. This review emphasizes the importance of continuous reality checks of experimental results obtained in vitro or with genetically modified animals with human disease.

Par3A is dispensable for the function of the glomerular filtration barrier of the kidney.

Polarity signaling through the atypical PKC (aPKC)-Par polarity complex is essential for the development and maintenance of the podocyte architecture and the function of the glomerular filtration barrier of the kidney. To study the contribution of Par3A in this complex, we generated a novel Pard3 podocyte-specific knockout mouse model by targeting exon 6 of the Pard3 gene. Genetic deletion of Pard3a did not impair renal function, neither at birth nor later in life. Even challenging the animals did not result in glomerular disease. Despite its well-established role in aPKC-mediated signaling, Par3A appears to be dispensable for the function of the glomerular filtration barrier. Moreover, its homolog Pard3b, and not Pard3a, is the dominant Par3 gene expressed in podocytes and found at the basis of the slit diaphragm, where it partially colocalizes with podocin. In conclusion, Par3A function is either dispensable for slit diaphragm integrity, or compensatory mechanisms and a high redundancy of the different polarity proteins, including Par3B, Lgl, or PALS1, maintain the function of the glomerular filtration barrier, even in the absence of Par3A.

Prohibitin-2 Depletion Unravels Extra-Mitochondrial Functions at the Kidney Filtration Barrier.

Mitochondrial fusion is essential for maintenance of mitochondrial function and requires the prohibitin ring complex subunit prohibitin-2 (PHB2) at the mitochondrial inner membrane. Loss of the stomatin/PHB/flotillin/HflK/C (SPFH) domain containing protein PHB2 causes mitochondrial dysfunction and defective mitochondria-mediated signaling, which is implicated in a variety of human diseases, including progressive renal disease. Here, we provide evidence of additional, extra-mitochondrial functions of this membrane-anchored protein. Immunofluorescence and immunogold labeling detected PHB2 at mitochondrial membranes and at the slit diaphragm, a specialized cell junction at the filtration slit of glomerular podocytes. PHB2 coprecipitated with podocin, another SPFH domain-containing protein, essential for the assembly of the slit diaphragm protein-lipid supercomplex. Consistent with an evolutionarily conserved extra-mitochondrial function, the ortholog of PHB2 in Caenorhabditis elegans was also not restricted to mitochondria but colocalized with the mechanosensory complex that requires the podocin ortholog MEC2 for assembly. Knockdown of phb-2 partially phenocopied loss of mec-2 in touch neurons of the nematode, resulting in impaired gentle touch sensitivity. Collectively, these data indicate that, besides its established role in mitochondria, PHB2 may have an additional function in conserved protein-lipid complexes at the plasma membrane.

Paul Ehrlich (1854-1915) and His Contributions to the Foundation and Birth of Translational Medicine.

Translational research and precision medicine are based on a profound knowledge of cellular and molecular mechanisms contributing to various physiologic processes and pathologic reactions in diverse organs. Whereas specific molecular interactions and mechanisms have been identified during the past 5 decades, the underlying principles were defined much earlier and originate from to the seminal observations made by outstanding researchers between 1850 and 1915. One of the most outstanding exponents of these scientists is Paul Ehrlich. His work resulted not only in the foundation and birth of modern hematology and immunology, but also led to the development of chemotherapy and specific targeted treatment concepts. In 2015, the Medical University of Vienna organized a memorial meeting, with the aim of honoring Paul Ehrlich's contributions to science, and to commemorate the 100th anniversary of his death. The authors of the current review served as faculty members and dedicate this paper to Paul Ehrlich and his remarkable contributions to medicine.