Lymphatic vessels in human adipose tissue.

Despite being considered present in most vascularised tissues, lymphatic vessels have not been properly shown in human adipose tissue (AT). Our goal in this study is to investigate an unanswered question in AT biology, regarding lymphatic network presence in tissue parenchyma. Using human subcutaneous (S-) and visceral (V-) AT samples with whole mount staining for lymphatic specific markers and three-dimensional imaging, we showed lymphatic capillaries and larger lymphatic vessels in the human VAT. Conversely, in the human SAT, microcirculatory lymphatic vascular structures were rarely detected and no initial lymphatics were found.

Recent advances and new insights into muscular lymphangiogenesis in health and disease.

The lymphatic vessels have been implicated in maintenance of interstitial fluid homeostasis and immune responses, and pathological conditions including inflammation, wound healing, lymphedema and tumor progression. The knowledge about the lymphatic structure and function in muscles, especially in muscular disorders, remains fragmentary and elusive. LYVE-1-positive initial lymphatics are generally found around skeletal muscle fibers, but not distributed in a fiber type-specific manner. Recent advances in lymphatic research have identified that exercise stimuli trigger adaptive changes in the behavior of initial lymphatics and upregulate lymphangiogenesis. Increasing evidence has supported that muscle disorders are closely correlated with lymphatic dysfunction, growth and remodeling. During these processes, VEGFR-3 and its ligands VEGF-C and VEGF-D are important regulators of muscular lymphangiogenesis. Further studies are necessary to clarify the structural and molecular plasticity of mammalian muscular lymphatics in response to endurance training and pathological events.

Orbital venous pattern in relation to extraorbital venous drainage and superficial lymphatic vessels in rats.

The purpose of this study was to demonstrate the normal and variant anatomy of extraorbital and intraorbital venous drainage together with retroorbital communication, and determine the lymphatic drainage from the superficial orbital region with a potential outlet of lymphatic vessel into the venous bloodstream. The study of the venous system was carried out on 32 Wistar rats by using corrosion casts methods and radiography, while the lymphatic system was studied in 12 Wistar rats following ink injection. Superficially, orbital veins are connected with extraorbital veins running through angular vein of the eye and the superficial temporal vein, and via the pterygoid plexus with the maxillary vein, which provide readily accessible communication routes in the spread of infection. The extent of intraorbital and periorbital venous drainage was ensured by the dorsal and ventral external ophthalmic vein through the infraorbital vein, which together formed the principal part of the ophthalmic plexus. Venous drainage of the eyeball was carried out mainly by the vortex veins, ciliary veins and internal ophthalmic vein. The highest variability, first presented by differences in structural arrangement and formation of anastomoses, was observed within the ventral external ophthalmic vein (22 cases) and the medial vortex vein (10 cases). Four vortex veins, one vein in each quadrant of the eye, were observed in rats. The vortex vein located on the ventral side of the eyeball was occasionally found as two veins (in four cases) in the present study. The lymphatic vessel from the lower eyelid entered into the mandibular lymph centre, and from the upper eyelid entered into the superficial cervical lymph centre, but both drained into the deep cranial cervical lymph node. The direct entry of lymph entering the veins without passing through lymph nodes was not observed.

Correlation of intratumoral lymphatic microvessel density, vascular endothelial growth factor C and cell proliferation in salivary gland tumors.

Lymphatic dissemination is one of the most important pathways for metastasis in many solid tumors, including head and neck carcinomas. The lymphatic growth of cancer has been used as a significant independent adverse prognostic factor and provides information about tumor progression. Salivary gland tumors present different prognoses and have the ability to develop metastases; however, this information regarding the lymphatic spread is scarce. This paper quantifies the lymphatic microvessel density (LMD) in benign and malignant salivary gland tumors and analyzes the relationship between LMD and tumor expression of vascular endothelial growth factors C (VEGF-C) and the proliferative index. The results show that there is no correlation between LMD, VEGF-C and the proliferative index in the majority of salivary gland tumors analyzed, apart from polymorphous low-grade carcinoma which exhibits statistical correlation between LMD and the proliferative index (p < 0.05). This correlation probably does not indicate a poor prognosis for this PLGA, since this is a low metastasizing carcinoma of the salivary glands. Different from other solid tumors, such as breast or prostatic carcinomas, there is no correlation between VEGF-C and LMD in salivary gland tumors, and so these traits are not able to estimate the metastatic risk or the prognosis of these tumors.

Modeling Proteolytically Driven Tumor Lymphangiogenesis.

With the exception of a limited number of sites in the body, primary tumors infrequently lead to the demise of cancer patients. Instead, mortality and a significant degree of morbidity result from the growth of secondary tumors in distant organs. Tumor survival, growth and dissemination are associated with the formation of both new blood vessels (angiogenesis) and new lymph vessels (lymphagenesis or lymphangiogenesis). Although intensive research in tumor angiogenesis has been going on for the past four decades, experimental results in tumor lymphangiogenesis began to appear only in the last 10 years. In this chapter we expand the models proposed by Friedman, Lolas and Pepper on tumor lymphangiogenesis mediated by proteolytically and un-proteolytically processed growth factors (Friedman and Lolas G, Math Models Methods Appl Sci 15(01):95-107, 2005; Pepper and Lolas G, Selected topics in cancer modeling: genesis, evolution, immune competition, and therapy. In: The lymphatic vascular system in lymphangiogenesis invasion and metastasis a mathematical approach. Birkhäuser Boston, Boston, pp 1-22, 2008). The variables represent different cell densities and growth factors concentrations, and where possible the parameters are estimated from experimental and clinical data. The results obtained from computational simulations carried out on the model equations produce dynamic heterogeneous ("anarchic") spatio-temporal solutions. More specifically, we observed coherent masses of tumor clusters migrating around and within the lymphatic network. Our findings are in line with recent experimental evidence that associate cluster formation with the minimization of cell loss favoring high local extracellular matrix proteolysis and thus protecting cancer invading cells from an immunological assault driven by the lymphatic network.

Observation of a Flowing Duct in the Abdominal Wall by Using Nanoparticles.

The primo vascular system (PVS) is being established as a circulatory system that corresponds to acupuncture meridians. There have been two critical questions in making the PVS accepted as a novel liquid flowing system. The first one was directly to show the flow of liquid in PVS and the second one was to explain why it was not observed in the conventional histological study of animal tissues. Flow in the PVS in the abdominal cavity was previously verified by injecting Alcian blue into a primo node. However, the tracing of the dye to other subsystems of the PVS has not been done. In the current work we injected fluorescent nanoparticles (FNPs) into a primo node and traced them along a primo vessel which was inside a fat tissue in the abdominal wall. Linea alba is a white middle line in the abdominal skin of a mammal and a band of fat tissue is located in parallel to the linea alba in the parietal side of the abdominal wall of a rat. In this fat band a primo vessel runs parallel to the prominent blood vessels in the fat band and is located just inside the parietal peritoneum. About the second question on the reason why the PVS was not in conventional histological study the current work provided the answer. Histological analysis with hematoxyline and eosine, Masson's trichrome, and Toluidine blue could not discriminate the primo vessel even when we knew the location of the PVS by the trace of the FNPs. This clearly explains why the PVS is hard to observe in conventional histology: it is not a matter of resolution but the contrast. The PVS has very similar structure to the connective tissues that surround the PVS. In the current work we propose a method to find the PVS: Observation of mast cell distribution with toluidine blue staining and the PN has a high density of mast cells, while the lymph node has low density.

Vascular Innervation in Benign Neurofibromas of Patients with Neurofibromatosis Type 1.

UNLABELLED: Neurofibroma constitutes a heterogeneous group of solid tumours occurring sporadically or in association with syndromes. The aspect of these peripheral nerve sheath tumours may vary considerably, with disseminated tumours covering various parts of the body or nodular/diffuse plexiform neurofibroma that can grow to an impressive size. Although neurofibromas have vascular density comparable to that of normal tissue, they have tendency to bleed upon surgery which is poorly understood. Herein we investigated whether this finding may result from alterations of peripheral vasculature innervation. Different types of neurofibroma and controls were evaluated with special reference to nerve fibre topography and vessel density. MATERIALS AND METHODS: Seventy-six formalin-fixed and paraffin-embedded tissue samples (63 neurofibromas and 13 skin biopsies) were retrieved from the archives of the Institute of Neuropathology, University Medical Center Hamburg-Eppendorf. Nerve fibres and blood vessels were differentiated immunohistochemically on 10-µm-thick tumour slices using antibodies against smooth muscle actin (arteries), protein gene product 9.5 (PGP9.5) and neurofilament (nerve fibres). Skin samples served as controls. Nerve fibre and vessel densities were quantified morphometrically. RESULTS: Nerve fibre density varied considerably. However, vascular innervation did not statistically significantly differ between the different tumour sub-groups and controls. Vessel density was not significantly increased in tumours compared to skin biopsies. Within the tumour sub-groups, diffuse plexiform neurofibroma presented a significantly higher vascular density than atypical neurofibroma (p=0.006). CONCLUSION: Blood vessel density and vascular innervation in the whole cohort of neurofibromas did not significantly differ from that of controls. Thus, the source of prolonged and intense bleeding of neurofibroma during surgical procedures cannot be explained by increased vessel density or loss of innervation, but may be attributed to other factors such as alterations in the structure of the vascular wall.

[TREATMENT OF POST-MASTECTOMY UPPER LIMB LYMPHEDEMA BY MODIFIED SIDE-TO-END LYMPHATICOVENULAR ANASTOMOSIS].

OBJECTIVE: To investigate the therapeutic effect of modified side-to-end lymphaticovenular anastomosis in the treatment of post-mastectomy upper limb lymphedema. METHODS: Between May 2010 and May 2011, 11 female patients with post-mastectomy upper limb lymphedema underwent a modified side-to-end lymphaticovenular anastomosis. The average age was 49.5 years (range, 38-55 years). Lymphedema occurred at 7-30 months (mean, 18.3 months) after resection of breast cancer, with an average disease duration of 25.5 months (range, 10-38 months). The left upper limb was involved in 5 cases and the right upper limb in 6 cases. In accordance with difference value between health and affected sides criteria, 5 cases were rated as moderate, and 6 cases as severe. RESULTS: Modified side-to-end lymphaticovenular anastomosis was successfully completed in all patients. Primary healing of incision was obtained in the other patients except 1 case of delayed healing. All patients were followed up for an average of 38.4 months (range, 36-40 months). Limb pain and swelling were relieved; no episodic attack or recurrence was observed. The circumference of affected upper arm was significantly decreased from preoperative (33.9 ± 3.7) cm to postoperative (31.0 ± 3.5) cm at 6 months and (30.9 ± 3.5) cm at 36 months (P < 0.05), but no significant difference was found between at 3 and 6 months (P > 0.05); the circumference of affected forearm was significantly decreased from preoperative (30.1 ± 3.6) cm to postoperative (27.8 ± 3.4) cm at 6 months and (27.7 ± 3.3) cm at 36 months (P < 0.05), but no significant difference was shown between at 6 and 36 months (P > 0.05). According to Campis evaluation standard to assess efficacy, the results were excellent in 3 cases, good in 6 cases, and improved in 2 cases CONCLUSIO: n Using modified side-to-end lymphaticovenular anastomosis may be effective in the treatment of upper limb lymphedema after mastectomy.

Lymphangiogenesis and Inflammation-Looking for the "Missing Pieces" of the Puzzle.

Several papers about lymphangiogenesis and inflammation focused on the detailed and complicated descriptions of the molecular pathways accompanying both non-tumor and tumor inflammatory-induced lymphatic vessel development. Many authors are tempted to present inflammatory-induced lymphangiogenesis in pathologic conditions neglecting the role of inflammatory cells during embryonic lymphatic vessel development. Some of the inflammatory cells are largely characterized in inflammatory-induced lymphangiogenesis, while others as mast cells, eosinophils, or plasma cells are less studied. No phenotypic characterization of inflammation-activated lymphatic endothelial cell is available in this moment. Another paradox is related to the existence of few papers regarding lymphangiogenesis inside lymphoid organs and for their related pathology. There are still several "missing pieces of such a big puzzle" of lymphangiogenesis and inflammation, with a direct impact on the ineffectiveness of the anti-inflammatory therapy as lymphangiogenesis inhibitors. The present paper will focus on the controversial issues of lymphangiogenesis and inflammation.

π-shaped lymphaticovenular anastomosis: the venous flow sparing technique for the treatment of peripheral lymphedema.

BACKGROUND: Nowadays, lymphaticovenular anastomosis has been recognized as an efficient microsurgical treatment for peripheral lymphedema. The technique based on two end-to-side anastomosis is named π-shaped lymphaticovenular anastomosis. This is the venous flow-sparing technique, in which the distal endothelial cells are not sacrificed. The purpose of this study is to evaluate the clinical results of π-shaped lymphaticovenular anastomosis in chronic lymphedema of the upper and lower limbs. PATIENTS AND METHODS: From November 2010 to August 2011, 20 patients with a peripheral lymphedema were treated by π-shaped lymphaticovenular anastomosis. A total of 12 patients had a lymphedema of the upper limb and 8 patients had a lymphedema of the lower limb. The mean age of the patients was 57.2 years (range, 44-78 years). The mean duration of lymphedema was 6.2 years (range, 1-23 years). The Campisi clinical stage range 2 to 5 (average, 3.3). Every patient was operated under local anesthesia. Four π-shaped lymphaticovenular anastomoses were performed per limb. RESULTS: The mean caliber of lymphatic vessels used for lymphaticovenular anastomosis was 0.55 mm (range, 0.3-0.8 mm). The mean caliber of subdermal venules was 1.2 mm (range, 0.5-2.1 mm).The average operative time to perform one π-shaped lymphaticovenular anastomosis was 55 minutes (range, 45-65 minutes). A venous backflow was found in 98 lymphaticovenular anastomosis (55.7%). Total 16 patients (80%) had a clinically significant circumferential reduction after surgery. The average volume differential reduction rate was 22.9% (range, 4.9-46.3) (p < 0.001). CONCLUSIONS: π-Shaped lymphaticovenular anastomosis is a supermicrosurgical method with a low morbidity to treat peripheral lymphedema. The procedure can easily be performed under local anesthesia, and the postoperative recovery is short. The results of this series demonstrate a clinical efficiency of the technique to reduce chronic lymphedema of the limbs.EBM level IV.

The clinical implication of cancer-associated microvasculature and fibroblast in advanced colorectal cancer patients with synchronous or metachronous metastases.

BACKGROUND: We aimed to evaluate the clinical significance of microvessel density (MVD), lymphatic vessel density (LVD), and cancer-associated fibroblasts (CAFs) in relation to tumor location in advanced colorectal cancer (CRC). METHODS: Using immunohistochemistry, we examined 181 advanced CRC patients for CD31 and D2-40 to measure MVD and LVD, respectively, α-smooth muscle actin (SMA) and desmin to identify CAFs, and PTEN to examine genetic changes of CAFs. To evaluate the regional heterogeneity of these properties, we examined tissue from four sites (the center and periphery of the primary cancer, a distant metastasis, and a lymph node metastasis) in each patient. RESULTS: MVD, LVD, and CAFs showed significant heterogeneity with respect to the tumor location. LVD was the greatest in the center of the primary cancers and the amount of CAFs was the lowest in distant metastases. In distant metastases, those from the lung had higher LVD and MVD, but fewer CAFs than those from the liver, peritoneum, or ovary. Patients with low MVD and LVD in the center of the primary cancer had worse outcomes and patients with few CAFs in distant metastases and in the primary tumor had a lower survival rate. PTEN expression in CAFs in distant metastases was lost in 11 of 181 CRC patients (6.1%), which was associated with a worse prognosis. CONCLUSIONS: The microenvironment, including cancer-associated microvasculature and fibroblasts, is heterogeneous with respect to the tumor location in CRC patients. Therefore, heterogeneity of microenvironments should be taken into account when managing CRC patients.

Aberrant expression of decoy receptor 3 in human breast cancer: relevance to lymphangiogenesis.

BACKGROUND: Decoy receptor 3 (DcR3), a decoy receptor against Fas ligand belonging to the tumor necrosis factor receptor superfamily, is overexpressed in some forms of cancer. It was recently reported that DcR3 could protect endothelial cells from apoptosis, implying a potential role in the development of vessels, whereas its role in the lymphangiogenesis remains unclear. In the present study, we studied the DcR3 expression and its relationship with the lymphatic microvessel density (LMVD) to investigate if it played a role in the lymph metastasis of human breast cancer. MATERIALS AND METHODS: Real-time polymerase chain reaction and immunohistochemistry were performed to measure the messenger RNA and protein expression of DcR3 in the breast cancer tissues, noncancerous counterparts, and axillary lymph node from 63 patients. LMVD in these specimens was assessed by counting the D2-40 labeled-microvessels. Furthermore, the correlations between DcR3 expression and LMVD and other clinicopathologic parameters were analyzed. RESULTS: DcR3 was overexpressed in the breast cancer tissue of 58 patients (92.1%) and was also expressed in vascular endothelial cells and tumor cells in the lymph nodes. LMVD in cancer tissue and lymph nodes were both positively correlated to the aberrant expression of DcR3. CONCLUSIONS: The relevance between DcR3 overexpression and LMVD revealed the existence of possible links between DcR3 and lymphangiogenesis. Based on these findings, it is important to further explore the regulation of lymphangiogenesis operated by the reverse tumor necrosis factor signaling of DcR3.

The prognostic implications of microvascular density and lymphatic vessel density in esophageal squamous cell carcinoma: Comparative analysis between the traditional whole sections and the tissue microarray.

Focal distribution of microvascular and lymphatic vessels is a critical issue in cancer, and is measured by tissue microarray (TMA) construction from paraffin-embedded surgically obtained tissues, a process that may not accurately reflect true focal distribution. The aim of this study was to assess the concordance of microvascular density (MVD) and lymphatic vessel density (LVD) in TMAs with corresponding whole sections, and to correlate the MVD or LVD with clinicopathological parameters in 124 cases of esophageal squamous cell carcinoma (ESCC). MVD, determined by CD105 immunohistochemistry of whole sections, was strongly associated with lymph node metastasis (p=0.000) and pTNM stage (p=0.001). Kaplan-Meier survival analysis showed that increasing CD105 microvessel count correlated with decreasing survival (p<0.001). The same result was acquired when MVD was calculated from tissue microarrays. Analysis of continuous data showed a highly significant correlation between whole sections and TMA data (Pearson r=0.522, p<0.001). Increasing LVD, as determined by D2-40 immunohistochemistry of whole sections, correlated with decreasing survival, but this relationship was undetectable using TMAs. In conclusion, we demonstrate that for the selected endothelial markers, TMAs can provide a realistic and reliable estimate of the extent of MVD, but not LVD in ESCC samples.

Embolization of the neck lymphatic varix, causing periodic neck swelling.

We report a case of a 44-year-old female patient, presented to us after years of recurrent intermittent episodes of unilateral left neck swelling. An MR lymphangiogram demonstrated a lymphatic varix at the confluence of the left upper extremity lymphatic ducts, confirmed by intranodal axillary lymphangiography. After successful catheterization of the feeding lymphatic vessels, the varix was successfully embolized with detachable microcoils and an autologous blood patch. The patient has been free from symptoms on subsequent outpatient follow-up.

Analysis of blood and lymph vascularization patterns in tissue-engineered human dermo-epidermal skin analogs of different pigmentation.

PURPOSE: Bioengineered dermo-epidermal skin analogs containing melanocytes represent a promising approach to cover large skin defects including restoration of the patient's own skin color. So far, little is known about the development of blood and lymphatic vessels in pigmented skin analogs after transplantation. In this experimental study, we analyzed the advancement and differences of host blood and lymphatic vessel ingrowth into light- and dark-pigmented human tissue-engineered skin analogs in a rat model. METHODS: Keratinocytes, melanocytes, and fibroblasts from light- and dark-pigmented skin biopsies were isolated, cultured, and expanded. For each donor, melanocytes and keratinocytes were seeded in ratios of 1:1, 1:5, and 1:10 onto fibroblast-containing collagen gels. The skin analogs were subsequently transplanted onto full-thickness wounds of immuno-incompetent rats and quantitatively analyzed for vascular and lymphatic vessel density after 8 and 15 weeks. RESULTS: The skin analogs revealed a significant difference in vascularization patterns between light- and dark-pigmented constructs after 8 weeks, with a higher amount of blood vessels in light compared to dark skin. In contrast, no obvious difference could be detected within the light- and dark-pigmented group when varying melanocyte/keratinocyte ratios were used. However, after 15 weeks, the aforementioned difference in blood vessel density between light and dark constructs could no longer be detected. Regarding lymphatic vessels, light and dark analogs showed similar vessel density after 8 and 15 weeks, while there were generally less lymphatic than blood vessels. CONCLUSION: These data suggest that, at least during early skin maturation, keratinocytes, melanocytes, and fibroblasts from different skin color types used to construct pigmented dermo-epidermal skin analogs have distinct influences on the host tissue after transplantation. We speculate that different VEGF expression patterns might be involved in this disparate revascularization pattern observed.

Signaling proteins are represented in tissue fluid/lymph from soft tissues of normal human legs at concentrations different from serum.

BACKGROUND: The mobile intercellular fluid flowing to and in the lymphatics contains filtered plasma products and substances synthesized and excreted by tissue cells. Among them are signaling proteins such as cytokines, chemokines, enzymes, and growth factors. They act locally in autocrine and paracrine systems regulating cell metabolism, proliferation, and formation of the ground matrix. They play an immunoregulatory role in infections, wound healing, and tumor cell growth. METHODS AND RESULTS: In this study we measured the concentration of selected cytokines, chemokines, tissue enzymes, and growth factors in tissue fluid/lymph drained from normal human leg soft tissues. Legs exposed to infections and trauma often result in development of lymphedema. Lymph was drained from superficial calf lymphatics using microsurgical techniques. Our studies showed generally higher concentrations of cytokines, chemokines, enzymes, and growth factors in lymph than in serum. The total protein L/S ratio was 0.22, whereas that of various lymph signaling proteins ranged between 1 and 10. CONCLUSIONS: This indicates that in addition to proteins filtered from blood, local cells contribute to lymph concentration by own production, depending on the actual cell requirement. Moreover, there were major individual differences of lymph levels with simultaneous stable serum levels. This suggests existence of a local autonomous regulatory humoral mechanism in tissues, not reflected in serum.

Clinicopathological significance of lymphangiogenesis and tumor lymphovascular invasion in indonesian breast cancers.

BACKGROUND: Lymphangiogenesis, assessed as lymphovascular density (LVD), is the initial step of generalized tumor lymphovascular invasion (LVI). It also involves VEGF-C as the most important protein family. Lymphangiogenesis among breast cancer cases correlations with several clinicopathological factors are important to determine prognosis and treatment strategies, but results have been controversial and require clarification. AIM: To define correlations between VEGF-C expression, LVD and LVI with several clinicopathological parameters from Indonesian breast cancer patients. MATERIALS AND METHODS: Using a cross-sectional study, a total of 48 paraffin-embedded tissues of breast cancer from Dr. Sardjito General Hospital Indonesia were assessed for VEGF-C expression, LVD and LVI by immunohistochemistry. Correlations of these markers with clinicopathological parameters like patient age, tumor size, lymph node status, grade, ER/PR and Her-2 status, cell proliferation and p-53 expression were investigated by linear analysis. Correlations of VEGF-C expression and LVI with several clinicopathological parameters were analyzed with Coefficient Contingency Chi-Square test. RESULTS: The mean of patients age was 53.0 year, pre and post-menopausal patients accounting for 56.3% and 43.8%, respectively. Some 10.4% were well, 41.7% moderate and 47.9% poorly differentiated. ER positivity was evident in 50% while PR and Her-2 positivity was found in 31.3% and 33.3%, respectively. Breast cancer cells with over-expression of p-53 was 64.6% and with high cell proliferation was 56.3%. Lymph node metastasis was noted in 63.5%, and LVI in 72.9%. Significant correlations were found between LVD and tumor size (p:0.037), grade (p:0.000), lymphnode status (p:0.036), LVI (p:0.003), as well as with p-53 and cell proliferation. There were also significant correlation of VEGF-C (p:0.011) and LVI (p:0.001) with tumor grade. Only ER status was found to have a correlation with tumor size (p:0.027). CONCLUSIONS: This study suggested that in Indonesian breast cancer patients, lymphangiogenesis is correlated with tumor size, grade, lymph node status and tumor lymphovascular invasion, the latter also being related with p-53 over expression and high cell proliferation.

In vitro inhibition of breast cancer spheroid-induced lymphendothelial defects resembling intravasation into the lymphatic vasculature by acetohexamide, isoxsuprine, nifedipin and proadifen.

BACKGROUND: As metastasis is the prime cause of death from malignancies, there is vibrant interest to discover options for the management of the different mechanistic steps of tumour spreading. Some approved pharmaceuticals exhibit activities against diseases they have not been developed for. In order to discover such activities that might attenuate lymph node metastasis, we investigated 225 drugs, which are approved by the US Food and Drug Administration. METHODS: A three-dimensional cell co-culture assay was utilised measuring tumour cell-induced disintegrations of the lymphendothelial wall through which tumour emboli can intravasate as a limiting step in lymph node metastasis of ductal breast cancer. The disintegrated areas in the lymphendothelial cell (LEC) monolayers were induced by 12(S)-HETE, which is secreted by MCF-7 tumour cell spheroids, and are called 'circular chemorepellent induced defects' (CCIDs). The putative mechanisms by which active drugs prevented the formation of entry gates were investigated by western blotting, NF-κB activity assay and by the determination of 12(S)-HETE synthesis. RESULTS: Acetohexamide, nifedipin, isoxsuprine and proadifen dose dependently inhibited the formation of CCIDs in LEC monolayers and inhibited markers of epithelial-to-mesenchymal-transition and migration. The migration of LECs is a prerequisite of CCID formation, and these drugs either repressed paxillin levels or the activities of myosin light chain 2, or myosin-binding subunit of myosin phosphatase. Isoxsuprine inhibited all three migration markers, and isoxsuprine and acetohexamide suppressed the synthesis of 12(S)-HETE, whereas proadifen and nifedipin inhibited NF-κB activation. Both the signalling pathways independently cause CCID formation. CONCLUSION: The targeting of different mechanisms was most likely the reason for synergistic effects of different drug combinations on the inhibition of CCID formation. Furthermore, the treatment with drug combinations allowed also a several-fold reduction in drug concentrations. These results encourage further screening of approved drugs and their in vivo testing.

Lymphatic vessel density as a prognostic marker in clinical stage I endocervical adenocarcinoma.

There are limited data evaluating the significance of lymphatic vessel density (LVD) as a prognostic marker in cervical adenocarcinoma. In this study, we investigated intratumoral and peritumoral LVD, using the lymphatic marker D2-40, as a prognostic marker in endocervical adenocarcinoma. Surgical specimens from 50 consecutive patients with endocervical adenocarcinoma treated with complete staging surgical procedures were reviewed. Selected tumor blocks were immunostained for D2-40 and CD31. Positively stained microvessels (MVs) were counted in densely vascular/lymphatic foci (hot spots) at 400x field in each specimen (0.17 mm). Results were expressed as the highest MV count identified within any single field. Both intratumoral CD31 MV and peritumoral D2-40 LVD showed significant correlation with depth of invasion (r=0.39, 0.37, respectively), percentage of circumferential involvement (r=0.36, 0.48, respectively), and lymphovascular invasion detected by D2-40 (r=0.45, 0.51, respectively; P<0.01). Only peritumoral D2-40 LVD showed a significant correlation with lymph node metastases (r=0.40; P<0.01), disease-free and overall survivals. Using univariate analysis, peritumoral D2-40 LVD showed significant correlation with lymphovascular invasion detected by D20-40 and lymph node metastases (P<0.05), which was maintained on multivariate analysis. D2-40 detected lymphovascular invasion in 16 of 50 (32%) cases, and showed a significant correlation with depth of invasion, lymph node metastases, involvement of parametrium (r=0.41, 0.38, 0.32, respectively; P<0.01), and disease-free survival. Our study showed that both angiogenesis and lymphangiogenesis play an important role in the progression of endocervical adenocarcinoma, and that peritumoral D2-40 LVD is an independent predictor of lymph node metastasis.

Blood supply to the lymphatic vessels in the leg: an incidental finding.

There are no reports or images of the blood supply to the lymphatic vessels. One lower limb of an unembalmed human cadaver was studied. Hydrogen peroxide (6%) was applied to find the lymphatic vessels by using a surgical microscope. The vessels were injected with a radio-opaque mixture and dissected. During the dissection, several sites of paralymphatics arteriole nutrient (PAN) vessels were found in close proximity to collecting lymphatic vessels in the medial aspect of the leg. The caliber of the lymphatic vessels was about 1 mm. The caliber of PAN vessels was <0.1 mm. The blood vessels were seen running along the lymphatic vessels. Some of them crossing the lymphatics and supplying the fatty tissue nearby and some running parallel on the lymph vessel walls. Histology sections show different-sized PAN vessels containing blood cells situated close to the lymphatic wall and within the lymphatic vessel wall. PAN vessels have been found and described. It will upgrade our anatomical knowledge and also be of benefit for medical and/or scientific research.