Lymphatic Collecting Vessels in Health and Disease: A Review of Histopathological Modifications in Lymphedema.

Secondary lymphedema of the extremities affects millions of people in the world as a common side effect of oncological treatments with heavy impact on every day life of patients and on the health care system. One of the surgical techniques for lymphedema treatment is the creation of a local connection between lymphatic vessels and veins, facilitating drainage of lymphatic fluid into the circulatory system. Successful results, however, rely on using a functional vessel for the anastomosis, and vessel function, in turn, depends on its structure. The structure of lymphatic collecting vessels changes with the progression of lymphedema. They appear initially dilated by excess interstitial fluid entered at capillary level. The number of lymphatic smooth muscle cells in their media then increases in the attempt to overcome the impaired drainage. When lymphatic muscle cells hyperplasia occurs at the expenses of the lumen, vessel patency decreases hampering lymph flow. Finally, collagen fiber accumulation leads to complete occlusion of the lumen rendering the vessel unfit to conduct lymph. Different types of vessels may coexist in the same patient but usually the distal part of the limb contains less affected vessels that are more likely to perform efficient lymphatic-venular anastomosis. Here we review the structure of the lymphatic collecting vessels in health and in lymphedema, focusing on the histopathological changes of the lymphatic vessel wall based on the observations on segments of the vessels used for lymphatic-venular anastomoses.

New Insights into the Pathophysiology of Primary and Secondary Lymphedema: Histopathological Studies on Human Lymphatic Collecting Vessels.

Background: Lymphedema is characterized by an accumulation of interstitial fluids due to inefficient lymphatic drainage. Primary lymphedema is a rare condition, including congenital and idiopathic forms. Secondary lymphedema is a common complication of lymph node ablation in cancer treatment. Previous studies on secondary lymphedema lymphatic vessels have shown that after an initial phase of ectasia, worsening of the disease is associated with wall thickening accompanied by a progressive loss of the endothelial marker podoplanin. Methods and Results: We enrolled 17 patients with primary and 29 patients with secondary lymphedema who underwent lymphaticovenous anastomoses surgery. Histological sections were stained with Masson's trichrome, and immunohistochemistry was performed with antibodies to podoplanin, smooth muscle α-actin (α-SMA), and myosin heavy chain 11 (MyH11). In secondary lymphedema, we found ectasis, contraction, and sclerosis vessel types. In primary lymphedema, the majority of vessels were of the sclerosis type, with no contraction vessels. In both primary and secondary lymphedema, not all α-SMA-positive cells were also positive for MyH11, suggesting transformation into myofibroblasts. The endothelial marker podoplanin had a variable expression unrelatedly with the morphological vessel type. Conclusions: Secondary lymphedema collecting vessels included all the three types described in literature, that is, ectasis, contraction, and sclerosis, whereas in primary lymphedema, we found the ectasis and the sclerosis but not the contraction type. Some cells in the media stained positively for α-SMA but not for MyH11. These cells, possibly myofibroblasts, may contribute to collagen deposition.

High-resolution MR lymphangiography for planning lymphaticovenous anastomosis treatment: a single-centre experience.

PURPOSE: This article illustrates the feasibility of MR lymphangiography (MRL) for imaging lymphatic vessels in patients with lymphedema, its accuracy in distinguishing lymphatic vessels from veins, and its utility for planning Lymphaticovenous anastomosis (LVA) treatment. MATERIALS AND METHODS: We prospectively enrolled 30 patients (24 women, range 18-70, 17 cases of lower limb lymphedema, 6 cases of primary lymphedema). All the patients underwent MRL, using a 1.5T MR unit (Signa Twin Speed Hdxt; GE), after the subcutaneous injection of gadobenate dimeglumine (Gd-BOPTA) with a little dose of lidocaine into the interdigital webs of the dorsal foot or hand. Lymphatic vessels identified for the LVA at MRL were histologically confirmed after surgery. Enhancement of lymphatic vessels and veins at different times after injection of contrast medium and their diameters were measured. RESULTS: A total of 79 lymphatic vessels were clearly identified in 29 patients at MRL; their morphology was tortuous in 22 patients and rectilinear in 7, whereas, the adjacent veins were straight with focal bulging only at the level of venous valve; the enhancement kinetic of the two different structures were different (p < 0.05) but the mean diameter of affected lymphatic vessels was similar to the adjacent veins (p > 0.05). Thirty-four out of 38 specimens of presumed lymphatic vessels at MRL, collected during surgery, resulted positive at the immunoistochemical marker d2-40, with a significant association (Chi-square = 40.421, DF = 1, p < 0.05, contingency coefficent 0.644). One patient had an early complication 1 month after treatment. CONCLUSIONS: MRL is easy and safe to use and combines extensive information on the anatomy and functionality of lymphatic vessels and veins in a single process; therefore, it could be useful in LVA treatment planning and evaluating possible super-microsurgical treatment complications in patients with lymphedema.

Local injection of bone marrow progenitor cells for the treatment of anal sphincter injury: in-vitro expanded versus minimally-manipulated cells.

BACKGROUND: Anal incontinence is a disabling condition that adversely affects the quality of life of a large number of patients, mainly with anal sphincter lesions. In a previous experimental work, in-vitro expanded bone marrow (BM)-derived mesenchymal stem cells (MSC) were demonstrated to enhance sphincter healing after injury and primary repair in a rat preclinical model. In the present article we investigated whether unexpanded BM mononuclear cells (MNC) may also be effective. METHODS: Thirty-two rats, divided into groups, underwent sphincterotomy and repair (SR) with primary suture of anal sphincters plus intrasphincteric injection of saline (CTR), or of in-vitro expanded MSC, or of minimally manipulated MNC; moreover, the fourth group underwent sham operation. At day 30, histologic, morphometric, in-vitro contractility, and functional analysis were performed. RESULTS: Treatment with both MSC and MNC improved muscle regeneration and increased contractile function of anal sphincters after SR compared with CTR (p < 0.05). No significant difference was observed between the two BM stem cell types used. GFP-positive cells (MSC and MNC) remained in the proximity of the lesion site up to 30 days post injection. CONCLUSIONS: In the present study we demonstrated in a preclinical model that minimally manipulated BM-MNC were as effective as in-vitro expanded MSC for the recovery of anal sphincter injury followed by primary sphincter repair. These results may serve as a basis for improving clinical applications of stem cell therapy in human anal incontinence treatment.

Systemic Sclerosis Sera Impair Angiogenic Performance of Dermal Microvascular Endothelial Cells: Therapeutic Implications of Cyclophosphamide.

In systemic sclerosis (SSc), dermal capillaries are progressively lost with consequent chronic tissue hypoxia insufficiently compensated by angiogenesis. Clinical studies reported that intravenous cyclophosphamide (CYC) may improve SSc-related peripheral microvascular damage. Recently, we showed that CYC treatment may normalize SSc sera-induced abnormalities in endothelial cell-matrix interactions. Our objective was to evaluate in vitro the effects of sera from treatment-naïve or CYC-treated SSc patients on dermal blood microvascular endothelial cell (dMVEC) angiogenesis, migration, proliferation and apoptosis. dMVECs were challenged with sera from 21 SSc patients, treatment-naïve (n = 8) or under CYC treatment (n = 13), and 8 healthy controls. Capillary morphogenesis on Geltrex matrix was significantly reduced upon challenge with sera from naïve SSc patients compared with healthy controls. When dMVECs were challenged with sera from CYC-treated SSc patients, their angiogenic capacity was comparable to that of cells treated with healthy sera. Wound healing capacity and chemotaxis in Boyden chamber were both significantly decreased in the presence either of naïve or CYC-treated SSc sera compared with healthy sera. WST-1 assay revealed that cell proliferation was significantly decreased in dMVECs challenged with sera from naïve SSc patients compared with healthy sera. Conversely, dMVEC proliferation was not impaired in the presence of sera from CYC-treated SSc patients. Accordingly, the percentage of TUNEL-positive apoptotic dMVECs was significantly higher in the presence of sera from naïve SSc patients than healthy controls, while CYC-treated SSc sera did not induce dMVEC apoptosis. Levels of the angiostatic mediators endostatin, pentraxin 3, angiostatin and matrix metalloproteinase-12 were all significantly elevated in sera from naïve SSc patients compared with sera from both healthy controls and CYC-treated SSc patients. In SSc, CYC treatment might boost angiogenesis and consequently improve peripheral microangiopathy through the normalization of the endothelial cell-matrix interactions, reduction of endothelial cell apoptosis and rebalance of dysregulated angiostatic factors.

Systemic sclerosis sera affect fibrillin-1 deposition by dermal blood microvascular endothelial cells: therapeutic implications of cyclophosphamide.

INTRODUCTION: Systemic sclerosis (SSc) is a connective tissue disorder characterized by endothelial cell injury, autoimmunity and fibrosis. The following three fibrillin-1 alterations have been reported in SSc. (1) Fibrillin-1 microfibrils are disorganized in SSc dermis. (2) Fibrillin-1 microfibrils produced by SSc fibroblasts are unstable. (3) Mutations in the FBN1 gene and anti-fibrillin-1 autoantibodies have been reported in SSc. Fibrillin-1 microfibrils, which are abundantly produced by blood and lymphatic microvascular endothelial cells (B-MVECs and Ly-MVECs, respectively), sequester in the extracellular matrix the latent form of the potent profibrotic cytokine transforming growth factor ß (TGF-ß). In the present study, we evaluated the effects of SSc sera on the deposition of fibrillin-1 and microfibril-associated glycoprotein 1 (MAGP-1) and the expression of focal adhesion molecules by dermal B-MVECs and Ly-MVECs. METHODS: Dermal B-MVECs and Ly-MVECs were challenged with sera from SSc patients who were treatment-naïve or under cyclophosphamide (CYC) treatment and with sera from healthy controls. Fibrillin-1/MAGP-1 synthesis and deposition and the expression of αvß3 integrin/phosphorylated focal adhesion kinase and vinculin/actin were evaluated by immunofluorescence and quantified by morphometric analysis. RESULTS: Fibrillin-1 and MAGP-1 colocalized in all experimental conditions, forming a honeycomb pattern in B-MVECs and a dense mesh of short segments in Ly-MVECs. In B-MVECs, fibrillin-1/MAGP-1 production and αvß3 integrin expression significantly decreased upon challenge with sera from naïve SSc patients compared with healthy controls. Upon challenge of B-MVECs with sera from CYC-treated SSc patients, fibrillin-1/MAGP-1 and αvß3 integrin levels were comparable to those of cells treated with healthy sera. Ly-MVECs challenged with SSc sera did not differ from those treated with healthy control sera in the expression of any of the molecules assayed. CONCLUSIONS: Because of the critical role of fibrillin-1 in sequestering the latent form of TGF-ß in the extracellular matrix, its decreased deposition by B-MVECs challenged with SSc sera might contribute to dermal fibrosis. In SSc, CYC treatment might limit fibrosis through the maintenance of physiologic fibrillin-1 synthesis and deposition by B-MVECs.

Morphometric analysis of intralobular, interlobular and pleural lymphatics in normal human lung.

In spite of their presumed relevance in maintaining interalveolar septal fluid homeostasis, the knowledge of the anatomy of human lung lymphatics is still incomplete. The recent discovery of reliable markers specific for lymphatic endothelium has led to the observation that, contrary to previous assumptions, human lymphatic vessels extend deep inside the pulmonary lobule in association with bronchioles, intralobular arterioles or small pulmonary veins. The aim of this study was to provide a morphometric characterization of lymphatic vessels in the periphery of the human lung. Human lung sections were immunolabelled with the lymphatic marker D2-40, followed by blood vessel staining with von Willebrand Factor. Lymphatic vessels were classified into: intralobular (including those associated with bronchovascular bundles, perivascular, peribronchiolar and interalveolar), pleural (in the connective tissue of the visceral pleura), and interlobular (in interlobular septa). The percentage area occupied by the lymphatic lumen was much greater in the interlobular septa and in the subpleural space than in the lobule. Most of the intralobular lymphatic vessels were in close contact with a blood vessel, either alone or within a bronchovascular bundle, whereas 7% were associated with a bronchiole and < 1% were not connected to blood vessels or bronchioles (interalveolar). Intralobular lymphatic size progressively decreased from bronchovascular through to peribronchiolar, perivascular and interalveolar lymphatics. Lymphatics associated with bronchovascular bundles had similar morphometric characteristics to pleural and interlobular lymphatics. Shape factors were similar across lymphatic populations, except that peribronchiolar lymphatics had a marginally increased roundness and circularity, suggesting a more regular shape due to increased filling, and interlobular lymphatics had greater elongation, due to a greater proportion of conducting lymphatics cut longitudinally. Unsupervised cluster analysis confirmed a marked heterogeneity of lymphatic vessels both within and between groups, with a cluster of smaller vessels specifically represented in perivascular and interalveolar lymphatics within the alveolar interstitium. Our data indicate that intralobular lymphatics are a heterogeneous population, including vessels surrounding the bronchovascular bundle analogous to the conducting vessels present in the pleural and interlobular septa, many small perivascular lymphatics responsible for maintaining fluid balance in the alveolar interstitium, and a minority of intermediate lymphatics draining the peripheral airways. These lymphatic populations could be differentially involved in the pathogenesis of diseases preferentially involving distinct lung compartments.

Human microvascular lymphatic and blood endothelial cells produce fibrillin: deposition patterns and quantitative analysis.

Fibrillin microfibrils constitute a scaffold for elastin deposition in the wall of arteries and form the anchoring filaments that connect the lymphatic endothelium to surrounding elastic fibers. We previously reported that fibrillin is deposited in a honeycomb pattern in bovine arterial endothelial cells, which also deposit microfibril-associated glycoprotein (MAGP)-1, whereas thoracic duct endothelial cells form an irregular web. The present immunohistochemical study was designed to verify whether lymphatic and blood human dermal microvascular endothelial cells (HDMECs) isolated from human foreskin by the sequential use of a pan-endothelial marker, CD31, and the lymphatic specific marker, D2-40, deposit fibrillin and MAGP-1. In both cell types, fibrillin and MAGP-1 co-localized and were deposited with different patterns of increasing complexity co-existing in the same culture. Fibrillin microfibrils formed a wide-mesh honeycomb leaving fibrillin-free spaces that were gradually filled. This modality of fibrillin deposition, similar to that of bovine large artery endothelial cells, was basically the same in blood and lymphatic HDMECs. In some lymphatic HDMECs, fibrillin was initially deposited as uniformly scattered short fibrillin strands probably as a result of anchoring filaments carried over from the vessels of origin. Our findings show that blood and lymphatic endothelial cells participate in fibrillin deposition in human skin.

Absence of lymphatic vessels in human dental pulp: a morphological study.

Few and controversial data are available in the literature regarding the presence of lymphatic vessels in the human dental pulp. The present study was designed to examine morphologically the existence of a lymph drainage system in human dental pulp. Human dental pulp and skin sections were immunohistochemically stained with specific antibodies for lymphatic endothelium (D2-40, LYVE-1, VEGFR-3 [vascular endothelial growth factor receptor-3], and Prox-1), with the pan-endothelial markers CD31 and von Willebrand factor (vWF), and with the blood-specific marker CD34. Several blood vessels were identified in human pulps and skin. Lymphatic vessels were found in all human skin samples but in none of the pulps examined. Western blotting performed on human dermis and on pulps treated with collagenase (to remove odontoblasts) confirmed these results. Transmission electron microscopy indicated that vessels which, by light microscopy, appeared to be initial lymphatic vessels had no anchoring filaments or discontinuous basement membrane, both of which are typical ultrastructural characteristics of lymphatic vessels. These results suggest that under normal conditions human dental pulp does not contain true lymphatic vessels. The various theories about dental pulp interstitial fluid circulation should be revised accordingly.

Lymphatic and blood vessels in scleroderma skin, a morphometric analysis.

Vascular involvement is frequent in systemic sclerosis, but the role of the lymphatic vasculature is poorly known. Our aim was to evaluate lymphatic vessels in systemic sclerosis skin lesions. We studied skin forearm biopsies of 9 patients with systemic sclerosis and 7 age-matched controls. Lymphatic vessels were labeled with the monoclonal antibody D2-40 and blood vessels with a polyclonal antibody to von Willebrand Factor. All blood and lymphatic vessels present in each section were counted and total area, inner luminal area, and shape factors were measured. The number of blood and lymphatic vessels in papillary dermis was greater and their diameter lower than in reticular dermis both in systemic sclerosis and controls. In the reticular dermis, the number of lymphatic vessels was markedly reduced in systemic sclerosis (4.9 +/- 1.1 microm(-2) versus 8.9 +/- 1.2 microm(-2)P = .03), and a similar trend was observed in papillary dermis (8.4 +/- 3.7 microm(-2) versus 8.1 +/- 5.3 microm(-2)). Interestingly, the number of periglandular lymphatics in systemic sclerosis was not different from controls. The inner luminal area (possibly indicating compensatory dilation) of lymphatic vessels, particularly the periglandular ones, was greater in systemic sclerosis than in controls. No differences were observed in the number of blood vessels, but the percentage of blood vessel profiles (without lumen) was significantly less in systemic sclerosis both in papillary and in reticular dermis. In conclusion, our data show that skin lesions in systemic sclerosis are characterized by a selective rarefaction of lymphatic vasculature that spares periglandular vessels and that might have a pathogenic role in the evolution and in the clinical manifestations of the disease.

Effects of morphine on testosterone levels in rat C6 glioma cells: modulation by anastrozole.

Rat C6 glioma cells are commonly used to investigate the functions of glial cells. To evaluate the presence of testosterone and its metabolism in rat C6 glioma cells, we cultured them in media with or without the addition of testosterone propionate and anastrozole, a blocker of aromatase, the enzyme needed to transform testosterone into estradiol. The same procedure was repeated with morphine (10 and 100 microM), known to decrease testosterone levels in the brain (in rats) and plasma (in rats and humans). Confluent cells were exposed to the test media for 48 h and then collected. Cell pellets were used to determine testosterone by radioimmunoassay. The C6 cells contained detectable levels of testosterone and the levels increased with the addition of testosterone to the medium. Aromatase blockage by anastrozole increased cellular levels of testosterone regardless of the addition of exogenous testosterone. Both concentrations of morphine dose-dependently decreased testosterone levels in the C6 cells; this effect was also present with the contemporary administration of anastrozole. Our findings show that testosterone is present in rat C6 glioma cells and can be metabolized by aromatase. Moreover, the presence of morphine in the culture medium strongly decreased testosterone, demonstrating that the glia would be a target of the morphine-induced hypogonadal effect.

The topography of microstructured surfaces differently affects fibrillin deposition by blood and lymphatic endothelial cells in culture.

While tissue-engineered blood vessels have already been successfully used in surgical practice, artificially restoring lymphatic circulation when needed is still far to be realized. Stability of arterial vessel wall depends on proper fibrillin deposition; fibrillin in fact is the scaffold for elastic fiber formation. In lymphatic vessels fibrillin is probably implied in lymph formation in response to interstitial requirements. This study was designed to verify whether fibrillin deposition is influenced by the topography of the substrate on which blood and lymphatic endothelial cells grow. Blood and lymphatic endothelial cells were cultured on microstructured surfaces with different topography: stripes of different widths (25, 50, and 100 microm), squares and rectangles, and spiral geometry, obtained by the photoimmobilization of Hyaluronan (Hyal) on aminosilanized glass. Cell orientation and fibrillin deposition were influenced by the topography of the microstructure. Blood endothelial cells deposited fibrillin as a bundle running parallel to the major axis of stripes and spirals, whereas the irregular network of fibrillin deposited by lymphatic endothelial cells was affected by the topography of the substrate only in the smallest stripes. These data bring a contribution to the basic knowledge required to design tissue-engineered blood and lymphatic vessels capable of adapting to the functional requirements of the surrounding environment.

Protein expression profiles of Bos taurus blood and lymphatic vessel endothelial cells.

The endothelium is a metabolically active organ that regulates the interaction between blood or lymph and the vessel or the surrounding tissue. Blood endothelium has been the object of many investigations whereas lymphatic endothelium biology is yet poorly understood. This report deals with a proteomic approach to the characterization and comparative analysis of lymphatic and blood vessel endothelial cells (ECs). By 2-DE we visualized the protein profiles of EC extracts from the thoracic aorta, inferior vena cava, and thoracic duct of Bos taurus. The three obtained electropherograms were then analyzed by specific software, and 113 quantitative and 25 qualitative differences were detected between the three endothelial gels. The cluster analysis of qualitative and quantitative differences evidenced the protein pattern of lymphatic ECs to be more similar to the venous than to the arterial one. Moreover, venous ECs were interestingly found showing a protein expression profile more similar to the lymphatic ECs than to the arterial ones. We also identified 64 protein spots by MALDI-TOF MS and ESI-IT MS/MS and three reference maps of bovine endothelium were obtained. The functional implications of the identified proteins in vascular endothelial biology are discussed.

Role of the Hyal-Cu (II) complex on bovine aortic and lymphatic endothelial cells behavior on microstructured surfaces.

The ability of micropatterned surfaces to modulate cell behavior is combined with the well-known angiogenic property of the hyaluronan-Cu (II) complex. Hyaluronan-Cu (II) microstripes 100 and 25 mum wide on aminosilanised glass substrates were fabricated by photoimmobilization following two different methods: i.e., method I consisting in the photoimmobilization of the Hyal-Cu (II) complex; and method II based on the photoimmobilization of Hyal followed by the coordination with Cu (II). The chemistry and topography of the fabricated micropatterned samples were investigated by ATR FT-IR, atomic absorption, AFM, SEM, and ToF-SIMS. ATR FT-IR analysis demonstrated that hyaluronan conjugated with a photoreactive moiety was able to coordinate Cu (II) ions and that the photoimmobilization process was successful, as indicated by the intensity decrease of the IR band of the azidic group after the photoreaction. AFM and SEM images showed that reproducible Hyal-Cu (II) microstructures with both chemical and topographical heterogeneities have been obtained by the two preparation methods. The distribution of copper on the fabricated Hyal-Cu (II) microstructures has been investigated by ToF-SIMS. In both ToF-SIMS images and spectra, on Hyal-Cu (II) microstructures prepared by method I, the Cu peak (63 m/z) was detected only on the Hyal-Cu (II) microstripes, while on Hyal-Cu (II) microstructures prepared by method II, the Cu peak showed the same intensity both on the Hyal-Cu (II) microstripes and on the aminosilanised glass substrate, in agreement with the higher amount of Cu revealed by atomic absorption. The influence of Hyal-Cu (II) micropatterned surfaces on BAEC and LEC, in terms of migration and adhesion, has been analyzed. The results obtained indicate that Hyal-Cu (II) influences BAEC behavior inducing cell migration, while it is devoid of any effect on LEC.

Lymphatics at the crossroads of angiogenesis and lymphangiogenesis.

The lymphatic system is implicated in interstitial fluid balance regulation, immune cell trafficking, oedema and cancer metastasis. However, the sequence of events that initiate and coordinate lymphatic vessel development (lymphangiogenesis) remains obscure. In effect, the understanding of physiological regulation of lymphatic vasculature has been overshadowed by the greater emphasis focused on angiogenesis, and delayed by a lack of specific markers, thereby limiting this field to no more than a descriptive characterization. Recently, new insights into lymphangiogenesis research have been due to the discovery of lymphatic-specific markers and growth factors of vascular endothelial growth factor (VEGF) family, such as VEGF-C and VEGF-D. Studies using transgenic mice overexpressing VEGF-C and VEGF-D have demonstrated a crucial role for these factors in tumour lymphangiogenesis. Knowledge of lymphatic development has now been redefined at the molecular level, providing an interesting target for innovative therapies. This review highlights the recent insights and advances into the field of lymphatic vascular research, outlining the most important aspects of the embryo development, structure, specific markers and methods applied for studying lymphangiogenesis. Finally, molecular mechanisms involved in the regulation of lymphangiogenesis are described.

Ameliorating effects of the immunomodulator 3-(2-ethylphenyl)-5-(3-methoxyphenyl)-1H-1,2,4-triazole in an experimental model of colitis in the rat.

The therapeutic efficacy of the immunomodulator 3-(2-ethylphenyl)-5-(3-methoxyphenyl)-1H-1,2,4-triazole (ST1959) in colonic inflammation was assessed in rats. One hour following colonic instillation of ethanolic 2,4,6-trinitrobenzene sulphonic acid (TNBS), intracolonic administration of 0.4 mg/kg ST1959 was started and continued once daily for 1 or 2 weeks. Daily administration of ST1959 for 1 week significantly reduced macroscopic and histological damage, myeloperoxidase activity, and colonic tissue levels of tumour necrosis factor-alpha and interferon-gamma. ST1959 did not affect interleukin-12 levels but significantly enhanced the production of interleukin-10 (sixfold increase). Two weeks of ST1959 treatment reduced the thickness of the colonic wall and myeloperoxidase activity to the same extent, and the histologic appearance of the mucosa was largely restored. The ameliorating effects seem to be ascribable to an impairment of both neutrophil infiltration/activation and tumour necrosis factor-alpha and interferon-gamma production, possibly consequent to the observed increase in the colonic tissue levels of the potent anti-inflammatory cytokine interleukin-10. Similar results were observed with the reference drug 5-aminosalycilic acid.

Lymphatic vessels in colorectal cancer and their relation with inflammatory infiltrate.

PURPOSE: The aim of this study was to determine why colorectal tumors confined to submucosa rarely metastasize. Under normal conditions, the submucosa contains many large lymphatic vessels with thin walls that would presumably favor the spread of cancer cells through the lymphatic system. METHODS: Specimens of colorectal cancer tissue, the border between tumor and normal tissue, and normal tissue were obtained from patients undergoing radical resection of colorectal cancer. The material was embedded in methacrylate resin for light microscopy and Epon for transmission electron microscopy examination. Light microscopy observations were routinely performed on serial sections. RESULTS: No lymphatic vessels were ever found in the tumor mass. The border area contained peritumoral inflammatory infiltrate of variable thickness. Where submucosal lymphatic vessels came into contact with peritumoral inflammatory infiltrate, they were profoundly altered: their endothelium was fragmented, and their walls were disrupted. These altered lymphatic vessels were almost always accompanied by mast cells, which were observed in the process of degranulating toward the lymphatic endothelium. No such alterations were detected in blood vessels. CONCLUSION: Our results suggest that mast cells, probably influenced by inflammatory infiltrate and/or colorectal cancer cells, destroy lymphatic vessels, which prevents cancer cells from spreading through the lymphatic system.

Focal adhesion molecules expression and fibrillin deposition by lymphatic and blood vessel endothelial cells in culture.

The microfibrils of anchoring filaments, a typical ultrastructural feature of initial lymphatic vessels, consist mainly of fibrillin and are similar to the microfibrils of elastic fibers. As we previously demonstrated, they radiate from focal adhesions of lymphatic endothelium to the perivascular elastic network. Although present in large blood vessels, fibrillin microfibrils have never been detected in blood capillaries. Here we report immunohistochemical evidence that cultured bovine aortic and lymphatic endothelial cells express fibrillin microfibrils. These microfibrils form an irregular web in lymphatic endothelial cells, whereas in blood vessel endothelial cells they are arranged in a honeycomb pattern. Cultured lymphatic and blood vessel endothelial cells also produce focal adhesion molecules: focal adhesion kinase, vinculin, talin, and cytoskeletal beta-actin. Our data suggest that anchoring filaments of initial lymphatic vessels in vivo may be produced by endothelium. Through their connection with focal adhesions, they may form a mechanical anchorage for the thin wall of initial lymphatic vessels and a transduction device for mechanical signals from the extracellular matrix into biochemical signals in endothelial cells. The complex anchoring filaments-focal adhesions may control the permeability of lymphatic endothelium and finely adjust lymph formation to the physiological conditions of the extracellular matrix. The different deposition of fibrillin microfibrils in blood vessel endothelial cells may be related to the necessity of withstanding shear forces. Thus, in our opinion, differences in fibrillin deposition imply a different role of fibrillin in blood vessel and lymphatic endothelium.