MCU controls melanoma progression through a redox-controlled phenotype switch.

Melanoma is the deadliest of skin cancers and has a high tendency to metastasize to distant organs. Calcium and metabolic signals contribute to melanoma invasiveness; however, the underlying molecular details are elusive. The MCU complex is a major route for calcium into the mitochondrial matrix but whether MCU affects melanoma pathobiology was not understood. Here, we show that MCUA expression correlates with melanoma patient survival and is decreased in BRAF kinase inhibitor-resistant melanomas. Knockdown (KD) of MCUA suppresses melanoma cell growth and stimulates migration and invasion. In melanoma xenografts, MCUA_KD reduces tumor volumes but promotes lung metastases. Proteomic analyses and protein microarrays identify pathways that link MCUA and melanoma cell phenotype and suggest a major role for redox regulation. Antioxidants enhance melanoma cell migration, while prooxidants diminish the MCUA_KD -induced invasive phenotype. Furthermore, MCUA_KD increases melanoma cell resistance to immunotherapies and ferroptosis. Collectively, we demonstrate that MCUA controls melanoma aggressive behavior and therapeutic sensitivity. Manipulations of mitochondrial calcium and redox homeostasis, in combination with current therapies, should be considered in treating advanced melanoma.

Control of the extracellular matrix by hypoxic lymphatic endothelial cells: Impact on the progression of lymphedema?

BACKGROUND: Initial lymphatic vessels do not have a continuous basement membrane. Therefore, the ability of lymphatic endothelial cells (LECs) to produce extracellular matrix (ECM) has received little attention. Untreated lymphedema is a chronic disease that progresses to massive fibrosclerosis in advanced stages. Expansion of the intercellular space and fibrosclerosis cause hypoxia, which also affects the LECs. RESULTS: We studied the expression of genes in human LECs in vitro by RNA sequencing, analyzed the effects of hypoxia (1% O2 ) vs. normoxia (21% O2 ), and focused on ECM genes. LECs express fibrillin-1 and many typical components of a basement membrane such as type IV, VIII, and XVIII collagen, laminin ß1, ß2, and α4, perlecan, and fibronectin. Under hypoxia, we found significant upregulation of expression of genes controlling hydroxylation of procollagen (PLOD2, P4HA1), and also cross-linking, bundling, and stabilization of collagen fibrils and fibers. Also striking was the highly significant downregulation of elastin expression, whereas fibulin-5, which controls the assembly of tropoelastin monomers, was upregulated under hypoxia. In the dermis from genital lymphedema, we observed significant PLOD2 expression in initial lymphatics. CONCLUSIONS: Overall, hypoxia results in the picture of a dysregulated ECM production of LECs, which might be partly responsible for the progression of fibrosclerosis in lymphedema.

Immunofluorescence Studies on the Expression of the SARS-CoV-2 Receptors in Human Term Placenta.

Until September 2021, the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2; COVID-19) pandemic caused over 217 million infections and over 4.5 million deaths. In pregnant women, the risk factors for the need of intensive care treatment are generally the same as in the overall population. Of note, COVID-19-positive women deliver earlier than COVID-19-negative women, and the risk for severe neonatal and perinatal morbidity and mortality is significantly higher. The probability and pathways of vertical transmission of the virus from the pregnant woman to the fetus are highly controversial. Recent data have shown that 54 (13%) of 416 neonates born to COVID-19-positive women were infected. Here, we investigated term placentas collected before the SARS-CoV-2 pandemic and studied the main COVID-19 receptors angiotensin-converting enzyme 2 (ACE2), transmembrane protease serine subtype 2 (TMPRSS2), as well as neuropilin 1 (NRP1). We performed real-time PCR and immunofluorescence on cryosections in combination with markers for syncytiotrophoblast, endothelial cells, macrophages and stromal cells. The PCR studies showed expression of both the truncated delta form of ACE2, which does not bind the COVID-19 spike protein, and the long form. The ACE2 antibody used does not distinguish between the two forms. We did not observe expression of the canonical SARS-CoV-2 entry machinery on syncytio- and cytotrophoblast. ACE2 and TMPRSS2 are co-expressed in a subpopulation of stromal cells, which in part are CD68-positive macrophages. NRP1 is localized to endothelial cells. In sum, the term placenta is not an organ that directly favors vertical transmission of COVID-19; however, microtraumas and placentitis may weaken its barrier function.

Differential in vitro effects of targeted therapeutics in primary human liver cancer: importance for combined liver cancer.

The incidence of primary liver tumors, hepatocellular carcinoma (HCC), intrahepatic cholangiocellular carcinoma (ICC), and combined HCC/ICC (cHCC/CC) is increasing. For ICC, targeted therapy exists only for a small subpopulation of patients, while for HCC, Sorafenib and Lenvatinib are in use. Diagnosis of cHCC/CC is a great challenge and its incidence is underestimated, bearing the risk of unintended non-treatment of ICC. Here, we investigated effects of targeted inhibitors on human ICC cell lines (HUH28, RBE, SSP25), in comparison to extrahepatic (E)CC lines (EGI1, CCC5, TFK1), and HCC/hepatoblastoma cell lines (HEP3B, HUH7, HEPG2). Cells were challenged with: AKT inhibitor MK-2206; multikinase inhibitors Sorafenib, Lenvatinib and Dasatinib; PI3-kinase inhibitors BKM-120, Wortmannin, LY294002, and CAL-101; and mTOR inhibitor Rapamycin. Dosage of the substances was based on the large number of published data of recent years. Proliferation was analyzed daily for four days. All cell lines were highly responsive to MK-2206. Thereby, MK-2206 reduced expression of phospho(p)-AKT in all ICC, ECC, and HCC lines, which mostly corresponded to reduction of p-mTOR, whereas p-ERK1/2 was upregulated in many cases. Lenvatinib showed inhibitory effects on the two HCC cell lines, but not on HEPG2, ICCs and ECCs. Sorafenib inhibited proliferation of all cells, except the ECC line CCC5. However, at reduced dosage, we observed increased cell numbers in some ICC experiments. Dasatinib was highly effective especially in ICC cell lines. Inhibitory effects were observed with all four PI3-kinase inhibitors. However, cell type-specific differences were also evident here. Rapamycin was most effective in the two HCC cell lines. Our studies show that the nine inhibitors differentially target ICC, ECC, and HCC/hepatoblastoma lines. Caution should be taken with Lenvatinib and Sorafenib administration in patients with cHCC/CC as the drugs may have no effects on, or might even stimulate, ICC.

Cross contamination meets misclassification: Awakening of CHP-100 from sleeping beauty sleep-A reviewed model for Ewing's sarcoma.

A human cell line of neuroblastic tissue, which was believed to have been lost to science due to its unavailability in public repositories, is revived and reclassified. In the 1970s, a triple set of neuroblastoma (NB) cell lines became available for research as MYCN-amplified vs nonamplified models (CHP-126/-134 and CHP-100, respectively). Confusingly, CHP-100 was used in subsequent years as a model for NB and, since the 1990s, as a model for neuroepithelioma and later as a model for Ewing's sarcoma (ES), which inevitably led to non-reproducible results. A deposit at a bioresource center revealed that globally available stocks of CHP-100 were identical to the prominent NB cell line IMR-32 and CHP-100 was included into the list of misidentified cell lines. Now we report on the rediscovery of an authentic CHP-100 cell line and provide evidence of incorrect classification during establishment. We show that CHP-100 cells carry a t(11;22)(q24;q12) type II EWSR1-FLI1 fusion and identify it as a classic ES. Although the question of whether CHP-100 was a virtual and never existing cell line from the beginning is now clarified, the results of all relevant publications should be considered questionable. Neither the time of the cross-contamination event with IMR-32 is known nor was the final classification as a model for Ewing family of tumors available with an associated short tandem repeat profile. After a long road of errors and confusion, authentic CHP-100 is now characterized as a type II EWSR1-FLI1 fusion model 44 years after its establishment.

A missing distal complex of the external and internal anal sphincters: a macroscopic and histologic study using Japanese and German elderly cadavers.

The lower margin of the internal anal sphincter (IAS) is considered to lie on a J-shaped, subcutaneous part (SCP) of the external anal sphincter (EAS). The lower IAS is united with the J-shaped SCP to form a smooth-striated muscle complex. In the first part of this study, we ensured the presence of the J-shaped EAS in the lateral wall of the anal canal from 12 near-term fetuses. Second, in the lateral anal wall, the examination of the longitudinal section from 20 male and 24 female Japanese cadavers (72-95 years-old) demonstrated that the J-shaped EAS was lost in 15 (34%) due to the very small SCP. Third, we demonstrated that the J-shaped EAS was restricted in the latera anal wall using longitudinal histological sections of the anal canal from 11 male Japanese cadavers (75-89 years-old). Therefore, a site-dependent difference in the IAS-EAS configuration was evident. Finally, we compared a frequency of the lost J-shape between human populations using 10 mm-thick frontal slices from 36 Japanese and 28 German cadavers. The two groups of cadavers were compatible in age (a 0.2-years' difference in males). The macroscopic observations revealed that the J-shaped EAS was absent from 13 (36%) Japanese and six (20%) German specimens, suggesting that the SCP degeneration occurred more frequent in elderly Japanese than elderly German individuals (p < 0.05). The distal IAS-EAS complex seemed to push residual feces out of the anal canal at a transient phase from evacuation to closure. The absence might be the first sigh of anal dysfunction.

Left/right difference in the course and division of the pulmonary arterial branches in the lung upper lobe: A study using human embryos and early fetuses.

Although left/right differences in a configuration of the pulmonary artery (PA) and its branches are well known, there is little information as to when and how such differences are established. Examination of serial sagittal sections of 25 embryos and fetuses at 6-7 weeks of gestation demonstrated that, at O'Rahilly stages 18-20, the right earliest first branch of PA originated in the anterior side of the upper lobar bronchus and overlay the upper bronchi, in contrast to the left branch which was located posteriorly and constricted medially by the upper posterior bronchus B1 + 2b. The right earliest branch was most likely to correspond to the future superior trunk, while the left branch might be a lingual artery. At stages 21-23, the upper posterior parenchyma was still underdeveloped in the left lung, since the ductus arteriosus and the left common cardinal vein seemed to make the left upper thoracic cavity narrow. Conversely, in the right lung, the thick S2 seemed to require a double arterial supply from both the superior and inferior arterial trunks. On the left, A3 originated at the lung apex and took a long descending course along the lung anterior surface. This high position of A3 might soon be corrected by an increased volume of S3. Overall, in contrast to the lower and middle lobes, early-developed branches of the PA did not accompany upper segmental and subsegmental bronchi. A mechanism "differential growth" seemed to explain how to correct the fetal morphology to provide the adult morphology with variations.

Three-dimensional analysis of the segmental arrangement of lower lung lobes in human fetuses: is this arrangement a miniature version of adult morphology?

Knowledge of the lung segment system is essential for understanding human anatomy and has great clinical relevance. The arrangement of 11 segments, including the S* or subsuperior segment, and its individual variations, are considered to be the same in fetal and adult lungs. The present study assessed the topographical anatomy of lower segmental and subsegmental bronchi by computer-assisted three-dimensional imaging of serial sagittal sections of both lungs of 22 embryos and fetuses of gestational age 6-7 weeks (crown-rump length 15.0-28.5 mm). Long inferior courses of B8b (basal) and B10c (medial) were observed in sagittal sections of both lungs. B8a (lateral) and B10b (lateral) in the right lungs were consistently underdeveloped, with S9 occupying most of the lateral half of the lower lobe. In some samples, B6b (lateral) did not reach the lateral surface. The lateral dominance of S9 was also seen in the left lungs. Some B* candidates were present, but B7 candidates were absent. Lateral and posterior expansions of S6b, S8a and S10b to cover S9 were observed in additional midterm and near-term lung sections, indicating that the original S9 dominance was 'corrected' by an increase in lung volume. Delayed growth of the lower lateral subsegments might induce mechanical stress, resulting in aberrant notches or fissures, such as those separating an independent posterior lobe. The segmental arrangement of fetal lungs was not stable, but was altered over a long fetal period after the complete subsegmental division of the bronchi, except for the minor bronchi B* and B7.

Absence of lymphatic vessels in term placenta.

BACKGROUND: There has been debate about the existence of lymphatic vessels in placenta. Lymphatic endothelial cell (LEC) markers such as LYVE-1 and podoplanin/D2-40 have been found, although PROX1 has not been detected. The most reliable marker for LECs is the double staining for CD31 and PROX1, which has not been performed yet. METHODS: We studied three term placentas and dissected them into three areas: i.) basal plate area, ii.) intermediate area, and iii.) chorionic plate area. We used immunofluorescence single and double staining with antibodies against CD31, PROX1, LYVE-1, VEGFR-3, D2-40/PDPN, CD34, CCBE-1, and vimentin, as well as nested PCR, qPCR, Western blot and transmission electron microscopy (TEM). RESULTS: At TEM level we observed structures that have previously mistakenly been interpreted as lymphatics, however, we did not find any CD31/PROX1 double-positive cells in placenta. Absence of PROX1 was also noted by nested PCR, qPCR and Western blot. Also, LEC marker VEGFR-3 was expressed only in a small number of scattered leukocytes but was absent from vessels. The LEC marker D2-40/PDPN was expressed in most stromal cells, and the LEC marker LYVE-1 was found in a considerable number of stromal cells, but not in endothelial cells, which were positive for CD31, CD34, CCBE-1 and vimentin. Additionally, vimentin was found in stromal cells. CONCLUSIONS: Our studies clearly show absence of lymphatics in term placenta. We also show that the functional area of the mother's endometrium is not penetrated by lymphatics in term pregnancy.

PECAM-1 modulates liver damage induced by synergistic effects of TNF-α and irradiation.

The mechanisms of radiation-induced liver damage are poorly understood. We investigated if tumour necrosis factor (TNF)-α acts synergistically with irradiation, and how its activity is influenced by platelet endothelial cell adhesion molecule-1 (PECAM-1). We studied murine models of selective single-dose (25 Gy) liver irradiation with and without TNF-α application (2 µg/mouse; i.p.). In serum of wild-type (wt)-mice, irradiation induced a mild increase in hepatic damage marker aspartate aminotransferase (AST) in comparison to sham-irradiated controls. AST levels further increased in mice treated with both irradiation and TNF-α. Accordingly, elevated numbers of leucocytes and increased expression of the macrophage marker CD68 were observed in the liver of these mice. In parallel to hepatic damage, a consecutive decrease in expression of hepatic PECAM-1 was found in mice that received radiation or TNF-α treatment alone. The combination of radiation and TNF-α induced an additional significant decline of PECAM-1. Furthermore, increased expression of hepatic lipocalin-2 (LCN-2), a hepatoprotective protein, was detected at mRNA and protein levels after irradiation or TNF-α treatment alone and the combination of both. Signal transducer and activator of transcription-3 (STAT-3) seems to be involved in the signalling cascade. To study the involvement of PECAM-1 in hepatic damage more deeply, the liver of both wt- and PECAM-1-knock-out-mice were selectively irradiated (25 Gy). Thereby, ko-mice showed higher liver damage as revealed by elevated AST levels, but also increased hepatoprotective LCN-2 expression. Our studies show that TNF-α has a pivotal role in radiation-induced hepatic damage. It acts in concert with irradiation and its activity is modulated by PECAM-1, which mediates pro- and anti-inflammatory signalling.

WNT signaling, the development of the sympathoadrenal-paraganglionic system and neuroblastoma.

Neuroblastoma (NB) is a tumor of the sympathoadrenal system arising in children under 15 years of age. In Germany, NB accounts for 7% of childhood cancer cases, but 11% of cancer deaths. It originates from highly migratory progenitor cells that leave the dorsal neural tube and contribute neurons and glial cells to sympathetic ganglia, and chromaffin and supportive cells to the adrenal medulla and paraganglia. Clinically, histologically and molecularly, NBs present as extremely heterogeneous, ranging from very good to very poor prognosis. The etiology of NB still remains unclear and needs to be elucidated, however, aberrant auto- and paracrine embryonic cell communications seem to be likely candidates to initiate or facilitate the emergence, progression and regression of NB. The wingless-type MMTV integration site (WNT) family of proteins represents an evolutionary highly conserved signaling system that orchestrates embryogenesis. At least 19 ligands in the human, numerous receptors and co-receptors are known, which control not only proliferation, but also cell polarity, migration and differentiation. Here we seek to interconnect aspects of WNT signaling with sympathoadrenal and paraganglionic development to define new WNT signaling cues in the etiology and progression of NB.

Co-localization of lymphoid aggregates and lymphatic networks in nose- (NALT) and lacrimal duct-associated lymphoid tissue (LDALT) of mice.

BACKGROUND: The lymphatic vascular pattern in the head of mice has rarely been studied, due to problems of sectioning and immunostaining of complex bony structures. Therefore, the association of head lymphoid tissues with the lymphatics has remained unknown although the mouse is the most often used species in immunology. RESULTS: Here, we studied the association of nasal and nasolacrimal duct lymphatics with lymphoid aggregates in 14-day-old and 2-month-old mice. We performed paraffin sectioning of whole, decalcified heads, and immunostaining with the lymphatic endothelial cell-specific antibodies Lyve-1 and Podoplanin. Most parts of the nasal mucous membrane do not contain any lymphatics. Only the region of the inferior turbinates contains lymphatic networks, which are connected to those of the palatine. Nose-associated lymphoid tissue (NALT) is restricted to the basal parts of the nose, which contain lymphatics. NALT is continued occipitally and can be found at both sides along the sphenoidal sinus, again in close association with lymphatic networks. Nasal lymphatics are connected to those of the ocular region via a lymphatic network along the nasolacrimal duct (NLD). By this means, lacrimal duct-associated lymphoid tissue (LDALT) has a dense supply with lymphatics. CONCLUSIONS: NALT and LDALT play a key role in the immune system of the mouse head, where they function as primary recognition sites for antigens. Using the dense lymphatic networks along the NLD described in this study, these antigens reach lymphatics near the palatine and are further drained to lymph nodes of the head and neck region. NALT and LDALT develop in immediate vicinity of lymphatic vessels. Therefore, we suggest a causative connection of lymphatic vessels and the development of lymphoid tissues.

The lymphatic vascular system of the mouse head.

Histological studies of the lymphatic vascular system in adult mice are hampered because bones cannot be sectioned properly. Here, we decalcified the heads of 14-day-old mice, embedded them in paraffin and stained resultant serial sections with the lymphendothelial-specific antibodies Lyve-1 and Podoplanin. We show that the tissues with the highest lymphatic vascular density are the dermis and the oral mucous membranes. In contrast, the nasal mucous membrane is devoid of lymphatics, except for its most basal parts below the vomeronasal organ. The inferior nasal turbinate contains numerous lymphatics and is connected to the nasolacrimal duct (NLD), which is ensheathed by a dense network of lymphatics. The lymphatics of the eye lids and conjunctiva are connected to those of the inferior nasal turbinate. We suggest that cerebro-spinal fluid (CSF) can drain via the optic nerve and NLD lymphatics, whereas CSF drained via the Fila olfactoria into the nasal mucous membrane is used for moisturization of the respiratory air. Tongue, palatine and buccal mucous membranes possess numerous lymphatics, whereas the dental pulp has none. Lymphatics are present in the maxillary gland and close to the temporomandibular joint, suggesting the augmentation of lymph flow by chewing and yawning. Lymphatics can also be found in the dura mater and in the dural septae entering into deeper parts of the brain. Our findings are discussed with regard to CSF drainage and potential routes for ocular tumor dissemination.

De novo hem- and lymphangiogenesis by endothelial progenitor and mesenchymal stem cells in immunocompetent mice.

Cellular pro-angiogenic therapies may be applicable for the treatment of peripheral vascular diseases. Interactions between mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) may provide such a treatment option. With the exception of some studies in man, experiments have only been performed in immunodeficient mice and rats. We studied an immunocompetent syngeneic mouse model. We isolated MSCs from bone marrow and EPCs from the lung of adult C57/Bl.6 mice and co-injected them in Matrigel subcutaneously in adult C57/Bl.6 mice. We demonstrate development of both blood vessels and lymphatics. Grafted EPCs integrated into the lining of the two vessel types, whereas MSCs usually did not incorporate into the vessel wall. Injections of each separate cell type did not, or hardly, reveal de novo angiogenesis. The release of VEGF-A by MSCs has been shown before, but its inhibitors, e.g., soluble VEGF receptors, have not been studied. We performed qualitative and quantitative studies of the proteins released by EPCs, MSCs, and cocultures of the cells. Despite the secretion of VEGF inhibitors (sVEGFR-1, sVEGFR-2) by EPCs, VEGF-A was secreted by MSCs at bioavailable amounts (350 pg/ml). We confirm the secretion of PlGF, FGF-1, MCP-1, and PDGFs by EPCs/MSCs and suggest functions for VEGF-B, amphiregulin, fractalkine, CXCL10, and CXCL16 during MSC-induced hem- and lymphangiogenesis. We assume that lymphangiogenesis is induced indirectly by growth factors from immigrating leukocytes, which we found in close association with the lymphatic networks. Inflammatory responses to the cellular markers GFP and cell-tracker red (CMPTX) used for tracing of EPCs or MSCs were not observed. Our studies demonstrate the feasibility of pro-angiogenic/lymphangiogenic therapies in immunocompetent animals and indicate new MSC/EPC-derived angiogenic factors.

Maldevelopment of dermal lymphatics in Wnt5a-knockout-mice.

Maintenance of tissue homeostasis and immune surveillance are important functions of the lymphatic vascular system. Lymphatic vessels are lined by lymphatic endothelial cells (LECs). By gene micro-array expression studies we recently compared human lymphangioma-derived LECs with umbilical vein endothelial cells (HUVECs). Here, we followed up on these studies. Besides well-known LEC markers, we observed regulation of molecules involved in immune regulation, acetylcholine degradation and platelet regulation. Moreover we identified differentially expressed WNT pathway components, which play important roles in the morphogenesis of various organs, including the blood vascular system. WNT signaling has not yet been addressed in lymphangiogenesis. We found high expression of FZD3, FZD5 and DKK2 mRNA in HUVECs, and WNT5A in LECs. The latter was verified in normal skin-derived LECs. With immunohistological methods we detected WNT5A in LECs, as well as ROR1, ROR2 and RYK in both LECs and HUVECs. In the human, mutations of WNT5A or its receptor ROR2 cause the Robinow syndrome. These patients show multiple developmental defects including the cardio-vascular system. We studied Wnt5a-knockout (ko) mouse embryos at day 18.5. We show that the number of dermal lymphatic capillaries is significantly lower in Wnt5a-null-mice. However, the mean size of individual lymphatics and the LEC number per vessel are greater. In sum, the total area covered by lymphatics and the total number of LECs are not significantly altered. The reduced number of lymphatic capillaries indicates a sprouting defect rather than a proliferation defect in the dermis of Wnt5a-ko-mice, and identifies Wnt5a as a regulator of lymphangiogenesis.

The chick chorioallantoic membrane as an in vivo xenograft model for Burkitt lymphoma.

BACKGROUND: Burkitt lymphoma (BL) is an aggressive malignancy that arises from B-cells and belongs to the group of Non-Hodgkin lymphomas (NHL). Due to the lack of appropriate in vivo models NHL research is mainly performed in vitro. Here, we studied the use of the chick chorioallantoic membrane (CAM) for the generation of human BL xenograft tumors, which we compared with known characteristics of the human disease. METHODS: In order to generate experimental BL tumors, we inoculated human BL2B95 and BL2-GFP cells on the CAM. BL2B95 xenograft-tumors were grown for seven days and subsequently analyzed with transmission electron and immunofluorescence microscopy, as well as histological staining approaches. BL2-GFP cells were studied at regular intervals up to seven days, and their metastatic behavior was visualized with intravital immunofluorescence techniques. RESULTS: Xenografted BL2B95 cells formed solid tumors in the CAM model with a Ki67-index greater than 90%, preservation of typical tumor markers (CD10, CD19, CD20), a 'starry sky' morphology, production of agyrophilic fibers in the stroma, formation of blood and lymphatic vessels and lymphogenic dissemination of BL2B95 to distant sites. We identified macrophages, lymphocytes and heterophilic granulocytes (chick homolog of neutrophils) as the most abundant immune cells in the experimental tumors. BL2-GFP cells could be traced in real-time during their distribution in the CAM, and the first signs for their dissemination were visible after 2-3 days. CONCLUSIONS: We show that xenografted BL2B95 cells generate tumors in the CAM with a high degree of cellular, molecular and proliferative concord with the human disease, supporting the application of the CAM model for NHL research with a focus on tumor-stroma interactions. Additionally we report that BL2-GFP cells, grafted on the CAM of ex ovo cultured chick embryos, provide a powerful tool to study lymphogenic dissemination in real-time.

The relationship between the secondary vascular system and the lymphatic vascular system in fish.

New technologies have resulted in a better understanding of blood and lymphatic vascular heterogeneity at the cellular and molecular levels. However, we still need to learn more about the heterogeneity of the cardiovascular and lymphatic systems among different species at the anatomical and functional levels. Even the deceptively simple question of the functions of fish lymphatic vessels has yet to be conclusively answered. The most common interpretation assumes a similar dual setup of the vasculature in zebrafish and mammals: a cardiovascular circulatory system, and a lymphatic vascular system (LVS), in which the unidirectional flow is derived from surplus interstitial fluid and returned into the cardiovascular system. A competing interpretation questions the identity of the lymphatic vessels in fish as at least some of them receive their flow from arteries via specialised anastomoses, neither requiring an interstitial source for the lymphatic flow nor stipulating unidirectionality. In this alternative view, the 'fish lymphatics' are a specialised subcompartment of the cardiovascular system, called the secondary vascular system (SVS). Many of the contradictions found in the literature appear to stem from the fact that the SVS develops in part or completely from an embryonic LVS by transdifferentiation. Future research needs to establish the extent of embryonic transdifferentiation of lymphatics into SVS blood vessels. Similarly, more insight is needed into the molecular regulation of vascular development in fish. Most fish possess more than the five vascular endothelial growth factor (VEGF) genes and three VEGF receptor genes that we know from mice or humans, and the relative tolerance of fish to whole-genome and gene duplications could underlie the evolutionary diversification of the vasculature. This review discusses the key elements of the fish lymphatics versus the SVS and attempts to draw a picture coherent with the existing data, including phylogenetic knowledge.

The lymphatic vascular system: much more than just a sewer.

Almost 400 years after the (re)discovery of the lymphatic vascular system (LVS) by Gaspare Aselli (Asellius G. De lactibus, sive lacteis venis, quarto vasorum mesaraicorum genere, novo invento Gasparis Asellii Cremo. Dissertatio. (MDCXXIIX), Milan; 1628.), structure, function, development and evolution of this so-called 'second' vascular system are still enigmatic. Interest in the LVS was low because it was (and is) hardly visible, and its diseases are not as life-threatening as those of the blood vascular system. It is not uncommon for patients with lymphedema to be told that yes, they can live with it. Usually, the functions of the LVS are discussed in terms of fluid homeostasis, uptake of chylomicrons from the gut, and immune cell circulation. However, the broad molecular equipment of lymphatic endothelial cells suggests that they possess many more functions, which are also reflected in the pathophysiology of the system. With some specific exceptions, lymphatics develop in all organs. Although basic structure and function are the same regardless their position in the body wall or the internal organs, there are important site-specific characteristics. We discuss common structure and function of lymphatics; and point to important functions for hyaluronan turn-over, salt balance, coagulation, extracellular matrix production, adipose tissue development and potential appetite regulation, and the influence of hypoxia on the regulation of these functions. Differences with respect to the embryonic origin and molecular equipment between somatic and splanchnic lymphatics are discussed with a side-view on the phylogeny of the LVS. The functions of the lymphatic vasculature are much broader than generally thought, and lymphatic research will have many interesting and surprising aspects to offer in the future.

High-resolution mass spectrometric analysis of the secretome from mouse lung endothelial progenitor cells.

Recently, we isolated and characterized resident endothelial progenitor cells from the lungs of adult mice. These cells have a high proliferation potential, are not transformed and can differentiate into blood- and lymph-vascular endothelial cells under in vitro and in vivo conditions. Here we studied the secretome of these cells by nanoflow liquid chromatographic mass spectrometry (LC-MS). For analysis, 3-day conditioned serum-free media were used. We found 133 proteins belonging to the categories of membrane-bound or secreted proteins. Thereby, several of the membrane-bound proteins also existed as released variants. Thirty-five proteins from this group are well known as endothelial cell- or angiogenesis-related proteins. The MS analysis of the secretome was supplemented and confirmed by fluorescence activated cell sorting analyses, ELISA measurements and immunocytological studies of selected proteins. The secretome data presented in this study provides a platform for the in-depth analysis of endothelial progenitor cells and characterizes potential cellular markers and signaling components in hem- and lymphangiogenesis.

Resveratrol and related stilbene derivatives induce stress granules with distinct clearance kinetics.

Stress granules (SGs) are ribonucleoprotein functional condensates that form under stress conditions in all eukaryotic cells. Although their stress-survival function is far from clear, SGs have been implicated in the regulation of many vital cellular pathways. Consequently, SG dysfunction is thought to be a mechanistic point of origin for many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Additionally, SGs are thought to play a role in pathogenic pathways as diverse as viral infection and chemotherapy resistance. There is a growing consensus on the hypothesis that understanding the mechanistic regulation of SG physical properties is essential to understanding their function. Although the internal dynamics and condensation mechanisms of SGs have been broadly investigated, there have been fewer investigations into the timing of SG formation and clearance in live cells. Because the lifetime of SG persistence can be a key factor in their function and tendency toward pathological dysregulation, SG clearance mechanisms deserve particular attention. Here we show that resveratrol and its analogues piceatannol, pterostilbene, and 3,4,5,4'-tetramethoxystilbene induce G3BP-dependent SG formation with atypically rapid clearance kinetics. Resveratrol binds to G3BP, thereby reducing its protein-protein association valency. We suggest that altering G3BP valency is a pathway for the formation of uniquely transient SGs.