Endothelium Of Lymphatic Vessels Research Articles

Hyaluronic Acid as a LYVE-1 Receptor Ligand in the Lymphatic System of Healthy Human Skin.

Immunohistochemical detection of the LYVE-1 marker in healthy human full-thickness skin (the epidermis and the dermis) was carried out. LYVE-1 expression was found in the endothelium of lymphatic capillaries located in the papillary dermis, in the endothelium of larger lymphatic vessels of the reticular dermis, and in fibroblasts, which indicates their joint participation in hyaluronan metabolism. LYVE-1+ staining detected for the first time in cells of the stratum basale, the stratum spinosum, and the stratum granulosum of healthy human epidermis indicates their participation in hyaluronan metabolism and allows us to consider the spaces between epidermis cells as prelimphatics.

Effect of subchronic use of unsymmetrical dimethylhydrazine on contractile activity of isolated lymphatic vessels

Introduction. Unsymmetric dimethylhydrazine (UDMH, heptyl) is widely used as a rocket fuel. Heptyl belongs to especially dangerous substances (hazard class I). UDMH with chronic intragastric administration has a cardiotropic effect, which served as the basis for studying its effect on the contractile activity of lymphatic vessels, since they are part of the vascular system. In addition, lymphatic vessels are considered as objects similar in terms of parameters of phase contractile activity with the heart and having a certain commonality of regulatory mechanisms of cardiomyocytes and smooth muscle cells of lymphangions.
 Materials and methods. The toxic effect of UDMH was evaluated on a model of isolated rat lymphatic vessels with subchronic intragastric administration of heptyl for 28 days at doses of 0.02; 0.2; and 2 mg/kg.
 Results. The use of UDMH in all studied doses led to a decrease in the contractile activity of lymphatic vessels, which manifested itself in the suppression of the frequency and amplitude of contractions and, as a consequence, the integral indicator – minute productivity. At the same time, the tonic voltage increased dose-dependent. When assessing the state of the endothelium of lymphatic vessels using acetylcholine, a decrease (inversion) of the response of lymphangions to the vasodilator was revealed.
 Limitations of the study. The study was performed on male white rats and does not take into account gender differences in the effect of NDMG on lymphatic vessels. 
 Conclusion. As a result of the study, it was found that UDMH in doses of 0.02; 0.2; and 2 mg/kg causes inhibition of the pumping function of lymphatic vessels, due to a violation of the processes of both excitation and contraction in smooth muscle cells of lymphangions.

David Slays Goliath: Exceptional Efficacy of Oral Vinorelbine in Conquering Metastatic Finger Angiosarcoma

Abstract Angiosarcoma (AS) is a rare and aggressive disease originating from blood or lymphatic vessel endothelium, representing <1% of soft-tissue sarcomas. The prognosis is generally poor, with a 5-year survival rate of only 30%–50%. The recurrence rate is high, and metastasis occurs in about half of the cases. The National Comprehensive Cancer Network guidelines recommend anthracycline-based, gemcitabine-based, and paclitaxel regimens as preferred first-line treatments for metastatic AS. Vinorelbine, a vinca alkaloid, is listed as supportive care in AS for patients unsuitable for intensive chemotherapy or who are heavily treated. Although the efficacy of vinorelbine is modest, some studies suggest its synergistic effect with immunotherapy. In our case report, a patient with metastatic AS received frontline paclitaxel followed by pembrolizumab, but this treatment failed. However, a complete response (CR) was achieved after oral vinorelbine. Remarkably, the patient remains in CR for 4.5 years after discontinuing vinorelbine.

Epsin Endocytic Adaptor Proteins in Angiogenic and Lymphangiogenic Signaling.

Circulating vascular endothelial growth factor (VEGF) ligands and receptors are central regulators of vasculogenesis, angiogenesis, and lymphangiogenesis. In response to VEGF ligand binding, VEGF receptor tyrosine kinases initiate the chain of events that transduce extracellular signals into endothelial cell responses such as survival, proliferation, and migration. These events are controlled by intricate cellular processes that include the regulation of gene expression at multiple levels, interactions of numerous proteins, and intracellular trafficking of receptor-ligand complexes. Endocytic uptake and transport of macromolecular complexes through the endosome-lysosome system helps fine-tune endothelial cell responses to VEGF signals. Clathrin-dependent endocytosis remains the best understood means of macromolecular entry into cells, although the importance of non-clathrin-dependent pathways is increasingly recognized. Many of these endocytic events rely on adaptor proteins that coordinate internalization of activated cell-surface receptors. In the endothelium of both blood and lymphatic vessels, epsins 1 and 2 are functionally redundant adaptors involved in receptor endocytosis and intracellular sorting. These proteins are capable of binding both lipids and proteins and are important for promoting curvature of the plasma membrane as well as binding ubiquitinated cargo. Here, we discuss the role of epsin proteins and other endocytic adaptors in governing VEGF signaling in angiogenesis and lymphangiogenesis and discuss their therapeutic potential as molecular targets.

A novel portable immuno-device for the recognition of lymphatic vessel endothelial hyaluronan receptor-1 biomarker using GQD-AgNPrs conductive ink stabilized on the surface of cellulose.

Lymphatic vessel endothelium expresses various lymphatic marker molecules. LYVE-1, the lymphatic vessel endothelial hyaluronan (HA) receptor, a 322-residue protein belonging to the integral membrane glycoproteins which is found on lymph vessel wall and is completely absent from blood vessels. LYVE-1 is very effective in the passage of lymphocytes and tumor cells into the lymphatics. As regards cancer metastasis, in vitro studies indicate LYVE-1 to be involved in tumor cell adhesion. Researches show that, in neoplastic tissue, LYVE-1 is limited to the lymphovascular and could well be proper for studies of tumor lymphangiogenesis. So, the monitoring of LYVE-1 level in human biofluids has provided a valuable approach for research into tumor lymphangiogenesis. For the first time, an innovative paper-based electrochemical immune-platform was developed for recognition of LYVE-1. For this purpose, graphene quantum dots decorated silver nanoparticles nano-ink was synthesized and designed directly by writing pen-on paper technology on the surface of photographic paper. This nano-ink has a great surface area for biomarker immobilization. The prepared paper-based biosensor was so small and cheap and also has high stability and sensitivity. For the first time, biotinylated antibody of biomarker (LYVE-1) was immobilized on the surface of working electrode and utilized for the monitoring of specific antigen by simple immune-assay strategy. The designed biosensor showed two separated linear ranges in the range of 20-320 pg ml-1 and 0.625-10 pg ml-1, with the acceptable limit of detection (LOD) of 0.312 pg ml-1. Additionally, engineered immunosensor revealed excellent selectivity that promises its use in complex biological samples and assistance for biomarker-related disease screening in clinical studies.

Solitary ovarian cancer cells in the peritoneum: What happens below the surface?

BackgroundIn advanced epithelial ovarian cancer (EOC), the peritoneum is the primary site of disease recurrence which occurs in >75% of patients despite complete cytoreductive surgery (CRS) and chemotherapy. Macroscopically undetectable remaining cancer cells are deemed to be a source for recurrent disease. We investigated characteristics of occult disease in biopsies of macroscopically normal peritoneum during CRS. Materials and methodsWe included 14 patients with advanced stage high grade serous ovarian cancer (HGSOC). Eleven patients had received neoadjuvant chemotherapy (NACT) and three patients were chemotherapy naïve. Each patient underwent three study-related peritoneal biopsies: 1) of a metastasis, 2) adjacent to a metastasis and 3) at distance from metastases. Cryostat sections were immunohistochemically stained for PAX8 and PanCK as markers of EOC cells and for CD31 as a marker for vascular and lymphatic endothelium. The sections were analyzed semi-quantitatively. ResultsMacroscopically normal peritoneum showed solitary PAX8-positive cells adjacent to and at distance from metastases in all patients. Thirteen percent of these PAX8-positive cells were found to be attached to the mesothelium and are presumably spread through intra-abdominal fluid. Eighty-seven percent of the solitary PAX8-positive cells were found in the stroma underneath the mesothelium, of which 59% were firmly attached to endothelium and 33% were found in the stroma. In most cases, no sign of proliferation of the solitary cells was observed. Only a few clusters of PAX8-positive cells were found. Chemotherapy did not affect these results. ConclusionsSolitary PAX8-positive cells are present in the macroscopically healthy-looking peritoneum of all EOC patients investigated, irrespective of the distance to macroscopically-visible metastases and of previous treatment. The majority of these solitary cancer cells were attached to endothelium of capillaries, venules or lymphatic vessels. Their solitary character and lack of proliferation suggests a dormant state, which could explain why these cells are unaffected by neo-adjuvant chemotherapy.

BILATERAL PNEUMOTHORACES SECONDARY TO ANGIOSARCOMA OF THE SCALP

TOPIC: Lung Cancer TYPE: Medical Student/Resident Case Reports INTRODUCTION: Angiosarcoma is a rare and aggressive tumor that develops from the endothelium of lymphatic or blood vessels and typically originates in the head and neck. It is a disease of older adults and, despite aggressive therapy, has a notoriously poor prognosis. Metastatic disease is common to the liver, breast, bone, and skin with 20% of patients developing solid, cavitary and cystic pulmonary lesions. Secondary spontaneous pneumothorax (SSP) due to malignancy is rare and develops from spontaneous rupture of cystic lung lesions in angiosarcoma. CASE PRESENTATION: The patient is an 87-year-old woman with a past medical history of angiosarcoma of the scalp status-post surgical resection and radiation treatment with new metastases to the carotid lymph nodes and spleen. She presented to the emergency department (ED) in acute hypoxic respiratory failure (AHRF) with an arterial oxygen saturation of 82% despite oxygen supplementation and was hypotensive. Chest x-ray and CT chest both demonstrated large bilateral pneumothoraces. Placement of bilateral chest tube thoracostomy resulted in lung expansion and improvement in oxygen saturation and hemodynamic status. The patient underwent bilateral pleurodesis with doxycycline and removal of chest tubes 3 days later with no further complications and was subsequently discharged home. One month later the patient presented to the ED with AHRF secondary to reoccurrence of bilateral spontaneous pneumothoraces. After chest tube thoracostomy the patient was transferred to inpatient hospice. DISCUSSION: SSP is an uncommon complication of pulmonary metastasis accounting for <1% of all spontaneous pneumothoraces. Simultaneous bilateral SSP is an even rarer occurrence accounting for <1% of all cases of spontaneous pneumothoraces. Other reported causes of bilateral pneumothorax include sarcoidosis, radiation, trauma, iatrogenesis, and cystic lung disease. Based on literature review there have been <20 reports worldwide of bilateral pneumothorax caused by pulmonary metastatic angiosarcoma. Prognosis after pneumothorax is poor with mortality reported within several months. Once the disease has progressed to this point, it is important to involve palliative care and hospice to discuss prognosis and goals of care. CONCLUSIONS: No established systemic treatment exists for angiosarcoma despite sporadic reports of moderately effective chemotherapy. With the high metastatic potential of angiosarcoma and its predilection towards the lungs, along with the rarity of SSP (especially bilaterally), a high degree of suspicion and close monitoring is required when pulmonary lesions are noted on imaging in this group of patients. REFERENCE #1: Upadya A, Amoateng-Adjepong Y, Haddad RG. Recurrent bilateral spontaneous pneumothorax complicating chemotherapy for metastatic sarcoma. South Med J. 2003;96(8):821-823. REFERENCE #2: Jimenez, D., Antaki, J., & Kamangar, N. (2016). Metastatic Pulmonary Angiosarcoma Presenting With Bilateral Secondary Spontaneous Pneumothoraces. Journal of Intensive Care Medicine, 32(4), 292–296. doi:10.1177/0885066616683323. REFERENCE #3: Marie Christine Aubry. Chapter 29 - Pulmonary Involvement by Extrapulmonary Neoplasms. Editor(s): Dani S. Zander, Carol F. Farver. In The Foundations in Diagnostic Pathology Series, Pulmonary Pathology, Churchill Livingstone, 2008. Pages 622-635. ISBN 9780443067419. DISCLOSURES: No relevant relationships by Anas Ahmed, source=Web Response No relevant relationships by Thomas Donnelly, source=Web Response No relevant relationships by Joy Wang, source=Web Response No relevant relationships by Steven Young, source=Web Response No relevant relationships by Nicholas Zingas, source=Web Response

Peri-tumoral Lymphovascular density by antipodoplanin antibody D2-40, as a predictor of nodal metastasis in oral squamous cell carcinoma

BACKGROUND:&#x0D; The involvement of regional lymph nodes by cancer is a major determinant of prognosis in oral squamous cell carcinoma (OSCC) and is a significant predictor of patient survival. Peri-tumoral lymphangiogenesis is being studied as an important tool in predicting neck lymph node metastasis. Podoplanin, a transmembrane glycoprotein is expressed on lymphatic vessel endothelium and considered as a marker of lymphangiogenesis.  The high podoplanin expression has been associated with invasion, progression, and metastasis of oral cancer. D2-40, an anti-podoplanin monoclonal antibody, is a specific marker of lymphatic endothelial cells. The present study was designed to investigate the peritumoral lymphovascular density (PT-LVD) by evaluating D2-40 expression and its potential utility in predicting lymph node metastasis in OSCC.&#x0D; METHODS: Seventy-two patients with OSCC were enrolled (36 with early and 36 with advanced-stage tumors). Differential expression of peritumoral lymphovascular density (PT-LVD) was evaluated using D2-40 expression by immunohistochemistry (IHC). Expression was categorized as low and high, derived from the mean of a number of vessels per 10 high power fields in hot-spots. Statistical methods were employed to correlate expression with age, gender, tumor site, stage, and neck lymph node involvement. &#x0D; RESULTS: We observed a significant association between peritumoral lymphovascular density (PT-LVD) with clinical neck lymph node status (p=0.001) and tumor stage (p=0.033).  There was no significant correlation of D2-40 expression with age, gender, tumor site, T stage, and tumor differentiation.&#x0D; CONCLUSION:  PT-LVD is a positive predictor of neck lymph node metastasis. It has translational potential, along with other clinical parameters for decision making in the treatment of the neck in oral cancer.

SUN-LB37 Atypical Presentation of Primary Adrenal Angiosarcoma With Cushing’s Syndrome - a Case Report

Angiosarcoma is a rare aggressive malignancy originating from the endothelium of blood and lymphatic vessels, accounting for 1% of all tissue sarcomas. These tumors usually invade skin and soft tissue, however they may also invade visceral organs and bones as metastatic disease. Approximately 50 cases have been reported in the literature describing primary adrenal angiosarcomas (PAA). This case represents the first report to describe concomitant Cushing’s syndrome in the setting of epithelioid PAA. A 38-year-old female with pre-diabetes mellitus and hyperlipidemia presented to the emergency department complaining of cough with bloody sputum. Physical exam was remarkable for bilateral lower extremity edema; no Cushing’s features appreciated. Bilateral subsegmental and lobar pulmonary emboli (PE) along with lytic lesions on the ribs were found on Computed Tomography (CT) PE protocol. Chest CT Angiography to prepare for arterial embolization to treat hemoptysis, demonstrated a large heterogeneous enhancing mass arising from the adrenal gland. MRI with adrenal protocol to evaluate incidentaloma revealed an indeterminate 5.2 x 5.6 x 5.2 cm right adrenal mass with central necrosis.Biochemical evaluation revealed a nonsupressible 1mg dexamethasone suppression test (3.3 ug/dL, ref: <1.8 ug/dL), slightly elevated urine metanephrines (191 ug/d, ref: 39 -143 ug/d) and normal plasma metanephrines (0.16 nmol/L, ref: 0.0 - 0.49 nmol/L). Given the biochemical evidence of mild hypercortisolism and uncertainty of etiology of other CT findings, the decision was made to pursue adrenalectomy to aid in the presumed oncologic diagnosis. She underwent successful adrenalectomy, however the postoperative course was complicated by hypotension resistant to fluids. ACTH stimulation test revealed a baseline cortisol <1.0 (ref: 0.0-9.0 ug/dL) with an ACTH of 8 pg/mL (ref: 6 to 58pg/mL), and 60 minute post stimulation cortisol level was 5.5 ug/dL (ref: >18.0 ug/dL). She was started on hydrocortisone replacement therapy, with clinical improvement in hypotension and was eventually tapered off steroids completely.Surgical pathology identified positive staining for CD31, CD34, factor 8, CAM 5.2, and Vimentin consistent with epithelioid angiosarcoma with lymphovascular invasion. There was additionally evidence of adrenocortical hyperplasia on the tissue resected. She established care with oncology and is currently completing 8 cycles of paclitaxel for metastatic disease to bones and lungs. Our patient’s pathology results, and postoperative course suggest the presence of concomitant non-ACTH dependent Cushing’s syndrome associated with epithelioid PAA. This case highlights the importance of thorough biochemical testing before adrenalectomy in the setting of PAA given the possibility of Cushing’s syndrome, which could contribute to post-surgical complications, and prolonged hospital stays.

Lymphatic Programing and Specialization in Hybrid Vessels

Building on a large body of existing blood vascular research, advances in lymphatic research have helped kindle broader investigations into vascular diversity and endothelial plasticity. While the endothelium of blood and lymphatic vessels can be distinguished by a variety of molecular markers, the endothelia of uniquely diverse vascular beds can possess distinctly heterogeneous or hybrid expression patterns. These expression patterns can then provide further insight on the development of these vessels and how they perform their specialized function. In this review we examine five highly specialized hybrid vessel beds that adopt partial lymphatic programing for their specialized vascular functions: the high endothelial venules of secondary lymphoid organs, the liver sinusoid, the Schlemm’s canal of the eye, the renal ascending vasa recta, and the remodeled placental spiral artery. We summarize the morphology and endothelial expression pattern of these vessels, compare them to each other, and interrogate their specialized functions within the broader blood and lymphatic vascular systems.

Inhibition of tumor formation and metastasis by a monoclonal antibody against lymphatic vessel endothelial hyaluronan receptor 1.

Although cancer metastasis is associated with poor prognosis, the mechanisms of this event, especially via lymphatic vessels, remain unclear. Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE‐1) is expressed on lymphatic vessel endothelium and is considered to be a specific marker of lymphatic vessels, but it is unknown how LYVE‐1 is involved in the growth and metastasis of cancer cells. We produced rat monoclonal antibodies (mAb) recognizing the extracellular domain of mouse LYVE‐1, and investigated the roles of LYVE‐1 in tumor formation and metastasis. The mAb 38M and 64R were selected from hybridoma clones created by cell fusion between spleen cells of rats immunized with RH7777 rat hepatoma cells expressing green fluorescent protein (GFP)‐fused mouse LYVE‐1 proteins and mouse myeloma cells. Two mAb reacted with RH7777 and HEK293F human embryonic kidney cells expressing GFP‐fused mouse LYVE‐1 proteins in a GFP expression‐dependent manner, and each recognized a distinct epitope. On immunohistology, the 38M mAb specifically stained lymphatic vessels in several mouse tissues. In the wound healing assay, the 64R mAb inhibited cell migration of HEK293F cells expressing LYVE‐1 and mouse lymphatic endothelial cells (LEC), as well as tube formation by LEC. Furthermore, this mAb inhibited primary tumor formation and metastasis to lymph nodes in metastatic MDA‐MB‐231 xenograft models. This shows that LYVE‐1 is involved in primary tumor formation and metastasis, and it may be a promising molecular target for cancer therapy.

Glucose transporter expression in the human colon.

AIMTo investigate by immunostaining glucose transporter expression in human colorectal mucosa in controls and patients with inflammatory bowel disease (IBD).METHODSColorectal samples were obtained from patients undergoing lower endoscopic colonoscopy or recto-sigmoidoscopy. Patients diagnosed with ulcerative colitis (n = 18) or Crohn’s disease (n = 10) and scheduled for diagnostic colonoscopy were enrolled. Patients who underwent colonoscopy for prevention screening of colorectal cancer or were followed-up after polypectomy or had a history of lower gastrointestinal symptoms were designated as the control group (CTRL, n = 16). Inflammatory status of the mucosa at the sampling site was evaluated histologically and/or endoscopically. A total of 147 biopsies of colorectal mucosa were collected and processed for immunohistochemistry analysis. The expression of GLUT2, SGLT1, and GLUT5 glucose transporters was investigated using immunoperoxidase labeling. To compare immunoreactivity of GLUT5 and LYVE-1, which is a marker for lymphatic vessel endothelium, double-labeled confocal microscopy was used.RESULTSImmunohistochemical analysis revealed that GLUT2, SGLT1, and GLUT5 were expressed only in short epithelial portions of the large intestinal mucosa. No important differences were observed in glucose transporter expression between the samples obtained from the different portions of the colorectal tract and between the different patient groups. Unexpectedly, GLUT5 expression was also identified in vessels, mainly concentrated in specific areas where the vessels were clustered. Immunostaining with LYVE-1 and GLUT5 antibodies revealed that GLUT5-immunoreactive (-IR) clusters of vessels were concentrated in areas internal to those that were LYVE-1 positive. GLUT5 and LYVE-1 did not appear to be colocalized but rather showed a close topographical relationship on the endothelium. Based on their LYVE-1 expression, GLUT5-IR vessels were identified as lymphatic. Both inflamed and non-inflamed mucosal colorectal tissue biopsies from the IBD and CTRL patients showed GLUT5-IR clusters of lymphatic vessels.CONCLUSIONGlucose transporter immunoreactivity is present in colorectal mucosa in controls and IBD patients. GLUT5 expression is also associated with lymphatic vessels. This novel finding aids in the characterization of lymphatic vasculature in IBD patients.

D2-40 Immunoreactivity of Lymphatic Vessels Endothelium and Representation of Lymphatic Vessels in the Liver of Human Fetuses of Different Gestational Age

Summary In the reference literature, there are a few studies on the development of the lymphatic system in the liver, especially human. This study aims to establish the presence, time of appearance, distribution and representation of expression D2-40 molecule – a marker of lymph vessels endothelial cells during the fetal period of the human liver development. The livers obtained from 20 human fetuses (10 male and 12 female), aged 12-37 gestational weeks, constituted our study material. Paraffin sections, 4 µm thick, were stained with hematoxylin and eosin for histological analysis, and with LSAB2/HRP method for immunohistochemistry using the D2-40 monoclonal antibody to mark lymphatic endothelial cells. The presence of lymphatic vessels was determined by morphometry, calculating their numerical and volume density. The study showed that expression of D2-40 molecule was absent in the liver lymphatic vessels in the first trimester of development, while in the second trimester intensive D2-40 immunoreactivity was observed in the lymph vessels of the liver capsule, and low D2-40 immunopositivity of the lymph vessels in large portal spaces. In the third trimester, intensive D2-40 immunoreactivity was observed in the lymph vessels of the liver capsule and in the endothelium of numerous lymphatic vessels of various shape and size, located in the smaller and larger portal areas. Volume and numerical density of lymphatic vessels in the portal areas of the liver during fetal development increased from the second to the third trimester of pregnancy, which was proportional to the increase in volume density of the hepatic portal spaces. Based on the obtained results, a conclusion may be drawn that the lymph vessels in the liver can be identified in the first half of the second trimester, and their number was growing proportionally by the end of pregnancy.

Immunohistochemical Heterogeneity of the Endothelium of Blood and Lymphatic Vessels in the Developing Human Liver and in Adulthood

The endothelium of liver sinusoids in relation to the endothelium of other blood vessels has specific antigen expression similar to the endothelium of lymphatic vessels. Bearing in mind that there is no consensus as to the period or intensity of the expression of certain antigens in the endothelium of blood and lymphatic vessels in the liver, the aim of our study was to immunohistochemically investigate the dynamic patterns of the expression of CD31, CD34, D2-40, and LYVE-1 antigens during liver development and in adulthood on paraffin tissue sections of human livers of 4 embryos, 38 fetuses, 6 neonates, and 6 adults. The results show that, in a histologically immature liver at the end of the embryonic period, CD34 molecules are expressed only on vein endothelium localized in developing portal areas, whereby the difference between portal venous branches and CD34-negative central veins belongs to the collecting venous system. In the fetal period, with aging, expression of CD34 and CD31 molecules on the endothelium of central veins and blood vessels of the portal areas increases. Sinusoidal endothelium shows light and sporadic CD34 immunoreactivity in the late embryonic and fetal periods, and is lost in the neonatal and adult periods, unlike CD31 immunoreactivity, which is poorly expressed in the fetal and neonatal periods but is present in adults. The endothelium of sinusoids and lymphatic vessels express LYVE-1, and the endothelium of lymphatic vessels express LYVE-1 and D2-40 but not CD34. Similarity between the sinusoidal and lymphatic endothelium includes the fact that both types are LYVE-1 positive and CD34 negative.

Low-molecular-weight hyaluronan (LMW-HA) accelerates lymph node metastasis of melanoma cells by inducing disruption of lymphatic intercellular adhesion

ABSTRACTEndothelial integrity defects initiate lymphatic metastasis of tumor cells. Low-molecular-weight hyaluronan (LMW-HA) derived from plasma and interstitial fluid was reported to be associated with tumor lymphatic metastasis. In addition, LMW-HA was proved to disrupt lymphatic vessel endothelium integrity, thus promoting lymphatic metastasis of tumor cells. Until now, there are few reports on how LMW-HA modulates lymphatic endothelial cells adhesion junctions and affects cancer cells metastasizing into lymph vessels. The aim of our study is to unravel the novel mechanism of LMW-HA in mediating tumor lymphatic metastasis. Here, we employed a melanoma metastasis model to investigate whether LMW-HA facilitates tumor cells transferring from foci to remote lymph nodes by disrupting the lymphatic endothelial integrity. Our data indicate that LMW-HA significantly induces metastasis of melanoma cells to lymph nodes and accelerates interstitial-lymphatic flow in vivo. Further experiments show that increased migration of melanoma cells across human dermal lymphatic endothelial cell (HDLEC) monolayers is accompanied by impaired lymphatic endothelial barrier function and increased permeability. The mechanism study reveals that VE-cadherin-β-catenin pathway and relevant signals are involved in modulating the interactions between endothelial cells and that a significant inhibition of lymphatic endothelium disruption is observed when antibodies to the LMW-HA receptor (LYVE-1) are present. Thus, our findings demonstrate a disruptive effect of LMW-HA on lymphatic endothelium continuity which leads to a promotion on melanoma lymphatic metastasis and also suggest a cellular signaling mechanism associated with VE-cadherin-mediated lymphatic intercellular junctions.

HISTOLOGICAL CHARACTERISTICS OF THE QUILTY EFFECT

Aim. Identifi cation of possible histological differences of the Quilty effect in acute rejection and its absence as well as studying the proliferation of blood and lymph vessels in the area of Quilty damage.Materials and methods. 883 endomyocardial biopsy materials from 352 patients were studied. Histological sections were stained with hematoxylin and eosin, Masson method; the endothelium of lymphatic vessels was stained with immunoperoxidase method using a marker D2-40.Results. The Quilty effect was observed both in acute rejection and inits absence. In the majority of cases the Quilty effect was of type «A» and it was combined with acute rejection. The Quilty effect of type «B» has been mostly in the G2R. Acute rejection is characterized by diffuse form of endocardium lymphoid infi ltration. Follicular form resembles lymphoid organ tissue. Different types and forms of the Quilty effect may be combined in the same biopsy. Proliferation of blood vessels presents in the area of the Quilty effect. Lymphatic vessels are missing in endocardium and in the area of the Quilty effect. They could be found only in the myocardium of endomiocardial biopsy.Conclusion. Diffuse lymphoid infi ltration of the endocardium is a characteristic feature of acute rejection of the transplanted heart. Follicular form of the Quilty effect is similar to lymphoid tissue whose role requires further study using immunohistochemical methods.

A Novel Model of Metastatic Conjunctival Melanoma in Immune-Competent Mice.

Conjunctival melanoma (CM) is an ocular surface tumor that can lead to fatal metastases. Patients developing, tumor-associated lymphangiogenesis have a significantly increased risk of metastatic disease, because tumor spread primarily occurs via lymphatic vessels to the draining lymph node. Here, we describe a novel immune-competent mouse model of CM that displays tumor-associated lymphangiogenesis with development of metastatic tumors. C57BL/6N mice received C57BL/6N-derived dermal melanoma cells (hepatocyte growth factor [HGF] cyclin dependent kinase-4 [Cdk4]+) or B16F10 via subconjunctival injection. A clinical score quantified primary tumor growth and metastases were identified by macroscopic examination of the draining lymph nodes, lung, and spleen. Confirmation of tumors and metastases was achieved by immunohistochemical staining for markers of pigmented cells (tyrosinase related protein-2 [TRP2]) and S-100, and of cell proliferation (Ki67). The intra- and peritumoral CD31+ blood and lymphatic vessel endothelium hyaluronan receptor-1 (LYVE-1)+ lymphatic vessels were quantified immunohistochemically. All mice rapidly developed aggressive TRP2+, S100+, and Ki67+ CM. Metastatic tumors were found in the lymph node (9%) and lung (6%) of HGF-Cdk4(R24C)-treated mice and in the spleen (8%) and lung (17%) of B16F10-treated mice. The amount of peri- and intratumoral blood vessels was significantly increased compared with lymphatic vessels. This CM model in immune-competent animals offers new possibilities to study the pathobiology of tumor growth, invasion, and mechanisms of metastatic tumor spread, and provides a robust model to explore new immune-based and antilymphangiogenic treatment modalities of this malignancy.

Tight junction protein claudin 4 in gastric carcinoma and its relation to lymphangiogenic activity

Background and study aimsGastric cancer is the second most common cause of cancer-related death worldwide. Claudins are a family of tight junction proteins that are biologically relevant in many cancer progression steps. This study aimed to investigate the expression of the intestinal claudin (claudin 4) in gastric carcinoma and to evaluate its relation to the different clinicopathologic prognostic parameters, especially lymphangiogenesis (production of new lymphatic vessels, measured by lymphovascular density (LVD)) and lymphovascular invasion (LVI). Patients and methodsFifty-five gastric carcinoma specimens were immunohistochemically stained for claudin 4 and D2-40 (for detection of lymphatic vessel endothelium). ResultsHigh expression of claudin 4 was detected in 26 of 55 (47.3%) cases. Low expression of claudin 4 was related to poorly differentiated type (p=0.001), non-intestinal (diffuse) type (p=0.001), deeper tumour invasion (p<0.001), lymph node metastasis (p=0.001), and higher stage (p=0.001). In addition, higher LVD was related to poorly differentiated types (p=0.001), non-intestinal type (p=0.001), lymph node metastasis (p=0.015), and higher tumour, node, metastasis (TNM) stage (p=0.001). LVI was related to lymph node metastasis (p=0.025), higher TNM stage (p=0.001), and LVD (p=0.001). Claudin 4 significantly correlated with both LVD (p=0.009) and LVI (p=0.009). ConclusionsHigh expression of claudin 4 was associated with the more differentiated intestinal-type gastric carcinoma and lost in poorly differentiated diffuse type. So, claudin 4 may be used as one of the differentiating markers between the two major types of gastric carcinoma (intestinal vs. diffuse). LVD and LVI were related to higher incidence of lymph node metastasis and therefore could be used as predictive markers for lymph node metastasis in limited specimens during early gastric carcinoma to determine the need for more invasive surgery. Low expression of claudin 4 was related to lymphangiogenesis. This may shed light on the relation of tight junction protein expression and lymphangiogenesis.

Neutrophils rapidly transit inflamed lymphatic vessel endothelium via integrin-dependent proteolysis and lipoxin-induced junctional retraction.

Neutrophils are the first leukocyte population to be recruited from the circulation following tissue injury or infection, where they play key roles in host defense. However, recent evidence indicates recruited neutrophils can also enter lymph and shape adaptive immune responses downstream in draining lymph nodes. At present, the cellular mechanisms regulating neutrophil entry to lymphatic vessels and migration to lymph nodes are largely unknown. Here, we have investigated these events in an in vivo mouse Mycobacterium bovis bacillus Calmette-Guérin vaccination model, ex vivo mouse dermal explants, and in vitro Transwell system comprising monolayers of primary human dermal lymphatic endothelial cells. We demonstrate that neutrophils are reliant on endothelial activation for adhesion, initially via E-selectin and subsequently, by integrin-mediated binding to ICAM-1 and VCAM-1, combined with CXCL8-dependent chemotaxis. Moreover, we reveal that integrin-mediated neutrophil adhesion plays a pivotal role in subsequent transmigration by focusing the action of matrix metalloproteinases and the 15-lipoxygenase-1-derived chemorepellent 12(S)-hydroxyeicosatetraenoic acid at neutrophil:endothelial contact sites to induce transient endothelial junctional retraction and rapid, selective neutrophil trafficking. These findings reveal an unexpectedly intimate collaboration between neutrophils and the lymphatic vessel endothelium, in which these phagocytic leukocytes act as pathfinders for their own transit during inflammation.

Hypoxia Induces Permeability and Giant Cell Responses of Andes Virus-Infected Pulmonary Endothelial Cells by Activating the mTOR-S6K Signaling Pathway

Andes virus (ANDV) is a South American hantavirus that causes a highly lethal hantavirus pulmonary syndrome (HPS) characterized by hypoxia, thrombocytopenia, and vascular leakage leading to acute pulmonary edema. ANDV infects human pulmonary microvascular and lymphatic endothelial cells (MECs and LECs, respectively) and nonlytically enhances the permeability of interendothelial cell adherence junctions in response to vascular endothelial growth factor (VEGF). Recent findings also indicate that ANDV causes the formation of giant endothelial cells. Here, we demonstrate that hypoxic conditions alone enhance permeability and giant cell responses of ANDV-infected MECs and LECs through activation of the mTOR signaling pathway. In contrast to infection of cells with nonpathogenic Tula virus (TULV), we observed that exposure of ANDV-infected MECs and LECs to hypoxic conditions resulted in a 3- to 6-fold increase in monolayer permeability and the formation of giant cells 3× to 5× normal size. ANDV infection in combination with hypoxic conditions resulted in the enhancement of hypoxia-inducible factor 1α (HIF1α)-directed VEGF A, angiopoietin 4, and EGLN3 transcriptional responses. Constitutive mTOR signaling induces the formation of giant cells via phosphorylation of S6K, and mTOR regulates hypoxia and VEGF A-induced cellular responses. We found that S6K was hyperphosphorylated in ANDV-infected, hypoxia-treated MECs and LECs and that rapamycin treatment for 1 h inhibited mTOR signaling responses and blocked permeability and giant cell formation in ANDV-infected monolayers. These findings indicate that ANDV infection and hypoxic conditions enhance mTOR signaling responses, resulting in enhanced endothelial cell permeability and suggest a role for rapamycin in therapeutically stabilizing the endothelium of microvascular and lymphatic vessels during ANDV infection.