Human collecting lymphatic glycocalyx identification by electron microscopy and immunohistochemistry.

Blood flow is translated into biochemical inflammatory or anti-inflammatory signals based onshear stress type, by means of sensitive endothelial receptors. Recognition of the phenomenon is of paramount importance for enhanced insights into the pathophysiological processes of vascular remodeling. The endothelial glycocalyx is a pericellular matrix, identified in both arteries and veins, acting collectively as a sensor responsive to blood flow changes. Venous and lymphatic physiology is interconnected; however, to our knowledge, a lymphatic glycocalyx structure has never been identified in humans. The objective of this investigation is to identify glycocalyx structures from ex vivo lymphatic human samples. Lower limb vein and lymphatic vessels were harvested. The samples were analyzed by transmission electron microscopy. The specimens were also examined by immunohistochemistry. Transmission electron microscopy identified a glycocalyx structure in human venous and lymphatic samples. Immunohistochemistry for podoplanin, glypican-1, mucin-2, agrin and brevican characterized lymphatic and venous glycocalyx-like structures. To our knowledge, the present work reports the first identification of a glycocalyx-like structure in human lymphatic tissue. The vasculoprotective action of the glycocalyx could become an investigational target in the lymphatic system as well, with clinical implications for the many patients affected by lymphatic disorders.

Prolonged Mechanical Circumferential Stretch Induces Metabolic Changes in Rat Inferior Vena Cava.

OBJECTIVE/BACKGROUND: Circumferential stretch on the vein wall has been suggested as a potential etiological factor in the development of varicose veins. However, the influence of vein wall stretch on vein metabolism has not yet been explored. The aim of this study was to investigate the effect of short and prolonged mechanical stretch on vein wall metabolism. METHODS: Circular segments of inferior vena cava from male Sprague-Dawley rats were exposed to normal 0.5-g (nonstretched) or high 2-g (stretched) tension for short (4 h) or prolonged (18 h) duration (five vein segments per group). Contraction response to phenylephrine (10-5 M) and KCl (96 mM) was elicited to observe the effect of circumferential stretch on vein function. The polar and organic metabolites in vein tissue were extracted using a bilayer extraction method. Aqueous and organic extracts were analyzed using nuclear magnetic resonance spectroscopy and ultra performance liquid chromatography coupled to mass spectrometry, respectively. Data acquired from both analytical platforms were analyzed using mathematical modeling. RESULTS: Increased concentrations of valine (p = .02) and choline (p = .03) metabolites and triglyceride moieties (p = .03) were observed in veins stretched for 18 h compared with the nonstretched/18 h group. DISCUSSION: Increased concentrations of branched chain amino acid valine and cell membrane constituent choline indicate increased muscle breakdown and increased metabolism of membrane phospholipids under stretch in an ex-vivo model. Increased intensities of triglyceride moieties in stretched vein segments for 18 h suggest that high pressure may induce an inflammatory response. CONCLUSION: This study has shown that prolonged mechanical circumferential stretch (18 h) alters the metabolic profile of rat inferior vena cava.

Glycosaminoglycan sulodexide modulates inflammatory pathways in chronic venous disease.

Inflammation represents an important epiphenomenon in the etiopathogenesis of chronic venous disease, a worldwide debilitating condition affecting millions of subjects. The pathophysiology of chronic venous disease (CVD) is based on the hemodynamic abnormalities in conjunction to alterations in cellular and extracellular matrix biocompounds. The endothelial dysfunction results from early perturbation in the endothelium linked to glycocalyx injury and promoted by inflammatory cells and mediators (such as matrix metalloproteinases and interleukins), which lead to progressive dilation of the vein resulting in chronic venous insufficiency. Activated leukocytes during the inflammatory process release enzymes, free radicals, chemokines and inflammatory cytokines in the vessel microenvironment, which are responsible for the changes of the venous wall and venous valve, reflux and venous hypertension, and the development/progression of tissue destruction and skin changes. Sulodexide, a highly purified mixture of glycosaminoglycans composed by 80% fast moving heparin and 20% of dermatan sulphate, exhibits anti-thrombotic and profibrinolytic properties, restoring also the essential endothelial glycocalyx. Glycosaminoglycan sulodexide has been also characterized to reduce the release of inflammatory cytokines/chemokines and to inhibit the matrix metalloproteinases-related proteolytic cascades, counteracting endothelial dysfunctions. The pleiotropic effects of sulodexide set the basis for a very promising agent in treating the spectrum of CVD.

Pathophysiology of chronic venous disease.

Chronic venous disease (CVD) is a debilitating condition with a prevalence between 60-70%. The disease pathophysiology is complex and involves genetic susceptibility and environmental factors, with individuals developing visible telengiectasias, reticular veins, and varicose veins. Patient with significant lower extremity symptoms have pain, dermal irritation, swelling, skin changes, and are at risk of developing debilitating venous ulceration. The signature of CVD is an increase in venous pressure referred to as venous hypertension. The various symptoms presenting in CVD and the clinical signs that are observed indicate that there is inflammation, secondary to venous hypertension, and it leads to a number of inflammatory pathways that become activated. The endothelium and glycocalyx via specialized receptors are critical at sensing changes in shear stress, and expression of adhesion molecules allows the activation of leukocytes leading to endothelial attachment, diapedisis, and transmigration into the venous wall/valves resulting in venous wall injury and inflammatory cells in the interstitial tissues. There is a complex of cytokines, chemokines, growth factors, proteases and proteinases, produced by activated leukocytes, that are expressed and unbalanced resulting in an environment of persistent inflammation with the clinical changes that are commonly seen, consisting of varicose veins to more advanced presentations of skin changes and venous ulceration. The structural integrity of protein and the extracellular matrix is altered, enhancing the progressive events of CVD. Work focusing on metabolic changes, miRNA regulation, inflammatory modulation and the glycocalyx will further our knowledge in the pathophysiology of CVD, and provide answers critical to treatment and prevention.

Inflammation in chronic venous ulcers.

Chronic venous ulcers (CVUs) occur in approximately 1% of the general population. Risk factors for chronic venous disease (CVD) include heredity, age, female sex and obesity. Although not restricted to the elderly, the prevalence of CVD, especially leg ulcers, increases with age. CVD has a considerable impact on health-care resources. It has been estimated that venous ulcers cause the loss of approximately two million working days and incur treatment costs of approximately $3 billion per year in the USA. Overall, CVD has been estimated to account for 1-3% of the total health-care budgets in countries with developed health-care systems. The pathophysiology of dermal abnormalities in CVU is reflective of a complex interplay that involves sustained venous hypertension, inflammation, changes in microcirculation, cytokine and matrix metalloproteinase (MMP) activation, resulting in altered cellular function and delayed wound healing.

Mechanisms of varicose vein formation: valve dysfunction and wall dilation.

Varicose veins are a common venous disease of the lower extremity. Although the mechanisms and determinants in the development of varicosities are not clearly defined, recent clinical studies and basic science research have cast some light on possible mechanisms of the disease. In varicose veins, there are reflux and incompetent valves as well as vein wall dilation. Primary structural changes in the valves may make them 'leaky', with progressive reflux causing secondary changes in the vein wall. Alternatively, or concurrently, the valves may become incompetent secondary to structural abnormalities and focal dilation in vein wall segments near the valve junctions, and the reflux ensues as an epiphenomenon. The increase in venous pressure causes structural and functional changes in the vein wall that leads to further venous dilation. Increase in vein wall tension augments the expression/activity of matrix metalloproteinases (MMPs), which induces degradation of the extracellular matrix proteins and affect the structural integrity of the vein wall. Recent evidence also suggests an effect of MMPs on the endothelium and smooth muscle components of the vein wall and thereby causing changes in the venous constriction/relaxation properties. Endothelial cell injury also triggers leukocyte infiltration, activation and inflammation, which lead to further vein wall damage. Thus, vein wall dilation appears to precede valve dysfunction, and the MMP activation and superimposed inflammation and fibrosis would then lead to chronic and progressive venous insufficiency and varicose vein formation.

Changes in cellular motility and cytoskeletal actin in fibroblasts from patients with chronic venous insufficiency and in neonatal fibroblasts in the presence of chronic wound fluid.

PURPOSE: Fibroblasts (fb) play an important role in wound healing involving motility, contraction, fibrosis, and expression of the cytoskeletal protein alpha-smooth muscle actin (alpha-sma). Patients with chronic venous insufficiency (CVI) are known to have dermal changes and impaired venous ulcer healing. To investigate whether these dermal-fb have an altered ability to migrate and whether chronic wound fluid from venous ulcers alters neonatal fb motility, we examined cell migration and alpha-sma. METHODS: Fibroblasts were cultured from the margin of venous ulcers (du-fb, n = 4, CEAP 6), from patients with venous reflux without ulcer (dr-fb, n = 5, CEAP 2), and from the ipsilateral thigh of the same patients with (pu-fb) and without (pr-fb) ulcer, respectively. The abbreviations used are p and d, which represent proximal and distal, respectively; u and r represent ulcer and reflux, respectively. Neonatal foreskin fibroblasts (nf-fb) were exposed to chronic venous ulcer wound fluid (CVUWF, 300 microg protein/mL, n = 3) or bovine serum albumin (BSA, control). Fibroblast motility was determined by means of time-lapse photo-images, and the rate (micrometer per hour) was calculated. Immunohistochemistry for alpha-sma was analyzed with confocal laser microscopy. RESULTS: The rate of motility (micrometer per hour +/- SEM) was decreased for both du-fb (11.4 +/- 0.7) and dr-fb (13.8 +/- 0.6), when compared with pu-fb (21.9 +/- 0.9) and pr-fb (24.7 +/- 1.1), respectively. The motility rate for nf-fb was lower in CVUWF (24.7 +/- 2.0) than in BSA (37.1 +/- 6.7). An elevated level of microfilament bundles of alpha-sma for both du-fb and dr-fb, compared with those of pu-fb and pr-fb, and also in nf-fb treated with CVUWF was demonstrated by means of immunohistochemistry. CONCLUSION: These data demonstrate a reduced motility in the dermal fb of patients with CVI. Patients with reflux disease without ulcer are predisposed to these changes. Furthermore, it appears that CVUWF causes changes in motility and alpha-sma expression in nf-fb as demonstrated in du-fb. These findings suggest that reduced motility and CVUWF, representing the microenvironment of venous ulcers, play a significant role in impaired wound healing.

The proliferative capacity of neonatal skin fibroblasts is reduced after exposure to venous ulcer wound fluid: A potential mechanism for senescence in venous ulcers.

PURPOSE: We have previously shown that fibroblasts cultured from venous ulcers display characteristics of senescence and have reduced growth rates. Susceptibility of young fibroblasts to the microcirculatory changes associated with venous ulcers, such as macrophage trapping and activation, could explain the prevalence of senescent fibroblasts in these wounds. METHODS: We tested the in vitro effect of venous ulcer wound fluid (VUWF), as well as pro-inflammatory cytokines known to be present in VUWF (TNF-alpha, IL-1beta, and TGF-beta1), on neonatal foreskin fibroblasts (NFFs). NFF growth rates, cellular morphology, and senescence-associated beta-galactosidase (SA-beta-Gal) activity were determined in the presence or absence of VUWF and the above cytokines. VUWF TNF-alpha concentration and the effect of anti-TNF-alpha antibody on VUWF inhibitory activity were determined in samples obtained from four patients with venous ulcers. RESULTS: NFF growth rates were significantly reduced by VUWF (42,727 +/- 6301 vs 3902 +/- 2191 P =. 006). TNF-alpha also significantly reduced NFF growth rates in a dose-dependent manner (P =.01). No significant growth-inhibitory activity was seen for IL-1alpha or TGF-beta. Incubation with VUWF significantly increased the percentage of SA-beta-Gal-positive fibroblasts in vitro on culture day 12 (P =.02). TNF-alpha and TGF-beta1 had similar effects. TNF-alpha was detected in all VUWF tested, with a mean of 254 +/- 19 pg/mL. CONCLUSION: These data suggest that the venous ulcer microenvironment adversely affects young, rapidly proliferating fibroblasts such as NFFs and induces fibroblast senescence. Pro-inflammatory cytokines such as TNF-alpha and TGF-beta1 might be involved in this process. The role of other unknown inhibitory mediators, as well as pro-inflammatory cytokines, in venous ulcer development and impaired healing must be considered.

The effect of passage number on fibroblast cellular senescence in patients with chronic venous insufficiency with and without ulcer.

BACKGROUND: Fibroblasts (fb) cultured from venous ulcer patients and patients with venous reflux disease without ulcer demonstrate characteristics of cellular senescence, such as increased fibronectin level and senescence-associated beta-galactosidase (SA beta-gal) positive cells. Cellular senescence is an in vitro event characterized by the progressive loss of proliferative capacity with increased passage number, and has been associated with impaired healing in vivo. This report examines progressive stages of cellular senescence in fb from the distal area (du-fb) and proximal fb (pu-fb) of patients with venous ulcer, as well as in distal fb (dr-fb) and proximal fb (pr-fb) from patients with venous reflux without ulcer, by comparing the population doubling time (T) and percent SA beta-gal expression. RESULTS: The mean value of T over 6 passages for fb in the ulcer group was 132.5 +/- 29.0 hours for pu-fb and 492.9 +/- 146.2 hours for du-fb (P = 0.0009). For fb in the reflux group the mean value of T over 5 passages was 79.3 +/- 12.8 hours for pr-fb and 94.2 +/- 16.8 hours for dr-fb (P = 0.8). Comparing ulcer and reflux fb, no difference in T was observed between pu-fb and pr-fb (P = 0.6), but a difference was noted between du-fb and dr-fb (P = 0.0004). The mean percent SA beta-gal activity for fb in the ulcer group was 11.2% +/- 3.1% for pu-fb and 63.8% +/- 8.9% for du-fb (P = 0.0001). Individual passages demonstrated significant difference (P <0.05) in SA beta-gal activity between pu-fb and du-fb at early and late passages. No difference was noted in SA beta-gal activity for fb in the reflux group or between pu-fb and pr-fb, but comparison between du-fb and dr-fb was significant (63.8% +/- 8.9% versus 7.8% +/- 2.9%; P = 0.0001). CONCLUSIONS: The in vitro passage of du-fb and pu-fb in chronic venous ulcer patients has an effect on T and cellular senescence as measured by SA beta-gal activity. Our data further suggest that du-fb are at a more progressive stage of cellular senescence when compared with pu-fb, and more importantly with fb cultured from patients with venous reflux without ulcer. These findings are consistent with impaired wound healing of venous stasis ulcer. The accumulation of senescent fb and a more advanced stage of cellular senescence of du-fb may explain why repeated episodes of venous ulceration are resistant to conservative treatment and require more aggressive measures of therapy.

Unilateral male breast masses: cancer risk and their evaluation and management.

Breast cancer is an uncommon cause of breast enlargement in the adult male. Overall, it accounts for <1 per cent of all male cancers. Although most male breast carcinomas are clinically apparent, distinguishing early breast cancer from gynecomastia, the most common cause of male breast enlargement, is considered a difficult task. To overcome this difficulty, many surgeons proceed directly to surgery as their initial diagnostic test. Although appropriate in some cases, the infrequent occurrence of male breast cancer and the diagnostic accuracy of mammography and fine-needle aspiration cytology suggest a modification of our present management. The aim of this study was to assess the incidence of breast cancer in men with unilateral breast masses and to propose a treatment algorithm for unilateral male breast masses. The medical records of 36 male patients who underwent subcutaneous mastectomy for a unilateral breast mass at the Buffalo Veterans Administration Medical Center between 1989 and 1996 were retrospectively reviewed. Data was collected on a standard data form. The median age was 63-years-old (range, 22-82). Gynecomastia was diagnosed in 30 patients (83%), lipoma in 4 patients (11%), invasive breast cancer in 1 patient (3%), and melanoma in situ in 1 patient (3%). Of the 30 patients with gynecomastia, 60% (18 patients) gave a history of a medical condition or use of medications known to cause gynecomastia, compared with 16 per cent (1 of 6) of the patients without gynecomastia (P = 0.08). Half of the patients with gynecomastia presented with an asymptomatic mass compared with 67 per cent of the patients without gynecomastia (P = not significant). The median duration of symptoms for patients with gynecomastia was 3 months. Men with unilateral breast masses have a low incidence of breast cancer. A male patient with a palpable unilateral breast mass consistent with gynecomastia on the basis of historical, physical and mammographic findings does not require surgical biopsy unless other clinical indications prevail. Lack of symptoms (pain) related to the mass is probably not helpful in deciphering gynecomastia from breast cancer.

The structure of streptonigrin semiquinone in solution.

Streptonigrin semiquinone (SQ.), a free radical intermediate implicated in the biological functioning of the antitumor antibiotic streptonigrin has been prepared and its structural properties in solution have been characterized. Through the use of electron paramagnetic resonance spectroscopy, the spin densities of the unpaired electron have been determined, indicating that the unpaired electron is largely confined to the quinolinesemiquinone moiety of the antibiotic. Unambiguous assignment of the hyperfine coupling constants was achieved employing isotopically labeled semiquinone radical, INDO molecular orbital calculations, and the study of unsubstituted 5,8-quinolinesemiquinone as a reference system. The assignments point to a negative spin density at the carbon para to the pyridine nitrogen in the radicals derived from both streptonigrin and the unsubstituted 5,8-quinolinequinone. Characterization of the properties of the streptonigrin semiquinone in solution indicate that the radical is stable in solution: it can be conveniently studied in 0.1 M methanolic lithium hydroxide or in aqueous organic solvent mixtures buffered with 0.06 M K3PO4 at pH 12.0. Under these conditions, the semiquinone shows completely reversible spectral changes between -10 to 60 degrees C. Lowering the pH from 12.0 to 7.0 in aqueous DMSO decreases the lifetime of the radical from two weeks to a few minutes. Changes in structural properties of streptonigrin semiquinone in solution have been found to occur mainly due to variation in solvation and freedom of rotation of the amino group. Decreasing the temperature of SQ. solution in methanol from 60 to -10 degrees C leads to an increase in the hyperfine coupling constant to the amino nitrogen from 1.28 to 1.40 G, and those of the two amino protons from 0.73 and 0.73 to 1.02 and 1.11 G respectively, while the other coupling constants change less than 3%. Greater electron spin delocalization onto the -NH2 group has been found throughout the solvent systems examined, yet the temperature at which