Antonella Rossi

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.

Lymphatic and blood vessels in scleroderma skin, a morphometric analysis

Summary 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 μm−2 versus 8.9 ± 1.2 μm−2P = .03), and a similar trend was observed in papillary dermis (8.4 ± 3.7 μm−2 versus 8.1 ± 5.3 μm−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.

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.

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 µM), 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. J. Cell. Physiol. 221: 1–4, 2009.

Micropatterned polysaccharide surfaces via laser ablation for cell guidance

Micropatterned materials were obtained by a controlled laser ablation of a photoimmobilised homogeneous layer of hyaluronic acid (Hyal) and its sulphated derivative (HyalS). The photoimmobilisation was performed by coating the polysaccharide, adequately functionalised with a photoreactive group, on aminosilanised glass substrate and immobilising it on the surface under UV light. Hyal or HyalS photoimmobilised samples were then subjected to laser ablation with wavelengths in the UV regions in order to drill the pattern. Four different patterns with stripes of 100, 50, 25 and 10 μm were generated. A chemical characterisation by attenuated total reflection/Fourier transform infrared (ATR/FT-IR) and time of flight-secondary ions mass spectrometry (TOF-SIMS) confirmed the success of the laser ablation procedure and the presence of alternating stripes of polysaccharide and native glass. The exact dimensions of the stripes were determined by atomic force microscopy. The analysis of cell behaviour in terms of adhesion, proliferation and movement using mouse fibroblasts (3T3 line) and bovine aortic endothelial cells (BAEC) was also performed.

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.

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

IntroductionSystemic 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 β (TGF-β). 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.MethodsDermal 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 αvβ3 integrin/phosphorylated focal adhesion kinase and vinculin/actin were evaluated by immunofluorescence and quantified by morphometric analysis.ResultsFibrillin-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 αvβ3 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 αvβ3 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.ConclusionsBecause of the critical role of fibrillin-1 in sequestering the latent form of TGF-β 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.

Systemic sclerosis sera affect fibrillin-1 deposition and focal adhesion molecule expression by dermal blood microvascular endothelial cells: therapeutic implications of cyclophosphamide

Systemic sclerosis (SSc, scleroderma) is characterized by fibrosis of the skin and internal organs, autoantibodies and diffuse microangiopathy. In SSc, fibrillin-1 microfibrils are disorganized and unstable. Mutations in FBN1 gene and anti-fibrillin- 1 autoantibodies have also been reported in SSc patients. Fibrillin-1 microfibrils sequester in the extracellular matrix the latent form of the potent pro-fibrotic cytokine transforming growth factor-β (TGF-β). We herein report 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 blood (B-MVECs) and lymphatic (Ly-MVECs) microvascular endothelial cells. Cells were challenged with sera from SSc patients, naive or under cyclophosphamide (CYC) treatment, and healthy controls. Fibrillin-1/MAGP-1 synthesis and deposition and the expression of αvβ3 integrin/ phosphorylated-FAK and vinculin/actin were evaluated by immunofluorescence and quantified by morphometric analysis. Fibrillin-1 and MAGP-1 co-localized 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 αvβ3 integrin expression significantly decreased upon challenge with sera from naive SSc patients compared with healthy controls. Vinculin was overexpressed in the cells exposed to the serum of naive patients with the diffuse form of SSc. B-MVECs challenged with sera from CYC-treated SSc patients showed fibrillin-1, αvβ3 integrin and vinculin levels comparable to those of controls. Ly-MVECs challenged with SSc sera did not differ from controls in the expression of any of the molecules assayed. Due to the critical role of fibrillin-1 in sequestering TGF-β in the extracellular matrix, its decreased deposition by B-MVECs challenged with SSc sera might contribute to dermal fibrosis. CYC treatment might limit fibrosis through the maintenance of physiologic fibrillin-1 synthesis and deposition by B-MVECs. CYC-induced normalization of αvβ3 integrin expression may restore effective interaction of endothelial cells with fibrillin. Since vinculin is necessary for angiogenesis, its normalization upon CYC treatment may contribute to restore the impaired angiogenesis found in SSc.

Morphometric analysis of lymphatics vessels in fibrotic human lung

In pulmonary fibrosis, the usual interstitial pneumonia (UIP) pattern is characterised by heterogeneous, patchy fibrosis, with areas of normal lung adjacent to areas of complete destruction (honeycombing) and by fibroblastic foci (FF). The NSIP pattern which is characteristic of systemic sclerosis, is characterised by a more homogeneous involvement of the lung without honeycombing and FF. Little is known on lymphatic vessels in lung fibrosis. Defective lymphatic clearance could lead to prolonged exposure to pathogenic antigens and/or pro-inflammatory/pro-fibrotic mediators. We evaluated the distribution and morphology of lymphatic vessels in lung biopsies of 6 patients with UIP, 6 NSIP and 5 controls. Consecutive sections were stained with Movat’s pentachrome and with double immunostaining for von Willebrand factor and podoplanin (D2-40). Area, perimeter and position were recorded for vessels with a diameter > 5µm. We investigated separately in lintralobular, sub-pleural, and interlobular spaces. Lymphatics were consistently larger in subpleural spaces and in interlobular septa than in intralobular tissue. In the latter, the density of lymphatic vessels was significantly reduced in NSIP and in UIP (both 21±1 mm-2) compared to controls (35±4 mm-2) . In controls, 85±6% of the intralobular lymphatics were close (< 100 µm) to a blood vessel, and only 5±4% were in the proximity of bronchoalveolar spaces, while in the disease groups they were less frequently perivascular (NSIP 55 ±3%, UIP 56 ±2%) and more frequently associated with the bronchoalveolar lumen (NSIP 85 ±3%, UIP 69 ±2%). By contrast, in interlobular septa, lymphatic density was significantly increased in NSIP (303±28 mm-2) and in UIP (286±124 mm-2) compared to controls (96±69 mm-2). No differences in lymphatic density was seen in subpleural spaces. Thus, our data show a marked redistribution of lymphatic vessels within the lung in pulmonary fibrosis, without noticeable differences between the NSIP and UIP patterns.