Daniela Ligi

Glycosaminoglycan Sulodexide Inhibition of MMP-9 Gelatinase Secretion and Activity: Possible Pharmacological Role Against Collagen Degradation in Vascular Chronic Diseases

We evaluated the effects of the glycosaminoglycan sulodexide (SDX; antithrombotic/profibrinolytic drug) on the activity and release of matrix metalloproteinases (MMPs) in human blood. This was a prospective non-randomized study, analyzing by zymography and ELISA the in vitro effects of SDX on pro-enzyme, complexed, and active MMP forms in plasma and serum from 60 healthy donors, and in U-937 leukemia cell line. The levels and zymographic profile of MMP-2 did not show significant changes among samples and during SDX treatments. However, pro- and complexed forms of MMP-9 were strongly affected by SDX treatment (P<0.001), with significant decrease of MMP-9 secretion from white blood cells in a dose-dependent fashion (P<0.0001), without any displacement of MMP prodomains. The mechanism of reduced release of MMP-9 forms from leukocytes and inhibition of proteolytic activity due to SDX treatment may support the hypothesis that drugs based upon inhibitors of MMP-9 activity may provide a therapeutic tool for the underlying pathological destruction of extracellular matrix, and offering novel pharmacologic applications for chronic inflammatory vascular diseases, including varicose vein and chronic venous diseases associated with enhanced MMP activation in blood and limbs.

Sulodexide Down-Regulates the Release of Cytokines, Chemokines, and Leukocyte Colony Stimulating Factors from Human Macrophages: Role of Glycosaminoglycans in Inflammatory Pathways of Chronic Venous Disease

Chronic venous disease (CVeD) is a debilitating condition that affects millions of individuals worldwide. The condition can result in varicose veins, or advance to severe skin changes and venous ulceration. The fundamental basis for CVeD is inflammation within the venous circulation and that it is subjected to increased hydrostatic pressure resulting in increased ambulatory venous pressure. The inflammation involves leukocytes, in particular macrophages and monocytes, inflammatory modulators and chemokines, cytokine expression, growth factors, metalloproteinase (MMP) activity, and many regulatory pathways that perpetuate inflammation. Sulodexide (SDX) is a glycosaminoglycan with pro-fibrinolytic and anti-thrombotic properties. We have previously demonstrated that SDX inhibits the secretion of pro-zymogen MMP-9 from human leukocytes without displacing high molecular complexes of MMP-9. The anti-inflammatory properties of SDX on activated leukocytes have not been well established. We hypothesized that SDX will reduce the secretion of inflammatory mediators from lipopolysaccharide (LPS)-stimulated macrophages. Therefore, we evaluated the effects of SDX on LPS-stimulated macrophage secretion of various inflammatory and anti-inflammatory cytokines, chemokines, and colony stimulating factors. We used microplatebased multiplex immunoassays. LPS-stimulated macrophages in vitro caused a substantial increase of interleukins, tumor necrosis factor, interferon, chemokines and colony stimulating factors. The addition of SDX caused both a dose-dependent and dose-independent decrease in nearly all of the inflammatory cytokines, chemokines and colony stimulating factors. These findings suggest that SDX has a significant effect on the release of inflammatory mediators from macrophages, and may be useful in the treatment of early and advanced CVeD.

Resolving breast cancer heterogeneity by searching reliable protein cancer biomarkers in the breast fluid secretome

BackgroundOne of the major goals in cancer research is to find and evaluate the early presence of biomarkers in human fluids and tissues. To resolve the complex cell heterogeneity of a tumor mass, it will be useful to characterize the intricate biomolecular composition of tumor microenvironment (the so called cancer secretome), validating secreted proteins as early biomarkers of cancer initiation and progression. This approach is not broadly applicable because of the paucity of well validated and FDA-approved biomarkers and because most of the candidate biomarkers are mainly organ-specific rather than tumor-specific. For these reasons, there is an urgent need to identify and validate a panel of biomarker combinations for early detection of human tumors. This is especially important for breast cancer, the cancer spread most worldwide among women. It is well known that patients with early diagnosed breast cancer live longer, require less extensive treatment and fare better than patients with more aggressive and/or advanced disease.ResultsIn the frame of searching breast cancer biomarkers (especially using nipple aspirate fluid mirroring breast microenvironment), studies have highlighted an optimal combination of well-known biomarkers: uPA + PAI-1 + TF. When individually investigated they did not show perfect accuracy in predicting the presence of breast cancer, whereas the triple combination has been demonstrated to be highly predictive of pre-cancer and/or cancerous conditions, approaching 97-100% accuracy.ConclusionDespite the heterogeneous composition of breast cancer and the difficulties to find specific breast cancer biomolecules, the noninvasive analysis of the nipple aspirate fluid secretome may significantly improve the discovery of promising biomarkers, helping also the differentiation among benign and invasive breast diseases, opening new frontiers in early oncoproteomics.

Omics profiles in chronic venous ulcer wound fluid: innovative applications for translational medicine

Chronic venous disease represents a healthcare problem due to high prevalence and recurrence rates. Studies on chronic venous ulcer wound fluid (CVUWF) have demonstrated increased inflammation and proteolysis which can cause tissue destruction and delayed healing. This review discusses: nearly all known metabolites discovered in the past 25 years in CVUWF studies; the omics approaches characterizing the microenvironment of human venous leg ulcers; and the use of biocompounds as prognostic biomarkers and as possible targets for therapeutic approaches. A biomarker is a biological compound that can be functional or non-functional, specific or non-specific in the diagnosis/prognosis to a disease state and may be quantified to determine progression or regression of disease. Omics studies in CVUWF provide the impetus for future identification of biomarkers within the intricate network in chronic venous disease and set the basis for determining the appropriate combination of molecules that are expressed with the healing status of venous leg ulcers.

Deciphering the single-cell omic: innovative application for translational medicine

Traditional technologies to investigate system biology are limited by the detection of parameters resulting from the averages of large populations of cells, missing cells produced in small numbers, and attempting to uniform the heterogeneity. The advent of proteomics and genomics at a single-cell level has set the basis for an outstanding improvement in analytical technology and data acquisition. It has been well demonstrated that cellular heterogeneity is closely related to numerous stochastic transcriptional events leading to variations in patterns of expression among single genetically identical cells. The new-generation technology of single-cell analysis is able to better characterize a cell’s population, identifying and differentiating outlier cells, in order to provide both a single-cell experiment and a corresponding bulk measurement, through the identification, quantification and characterization of all system biology aspects (genomics, transcriptomics, proteomics, metabolomics, degradomics and fluxomics). The movement of omics into single-cell analysis represents a significant and outstanding shift.

Chronic venous disease – Part II: Proteolytic biomarkers in wound healing

Venous leg ulcers (VLU) are characterized by sustained proteolytic microenvironment impairing the healing process. Wound fluid (WF) reflect the biomolecular activities occurring within the wound area; however, it is unclear if WF from different healing phases have different proteolytic profiles and how VLU microenvironment affects the wound healing mechanisms. We investigated the proteolytic network of WF from distinct VLU phases, and in WF- and LPS-stimulated THP-1 monocytes treated with glycosaminoglycan sulodexide, a well known therapeutic approach for VLU healing. WF were collected from patients with VLU during inflammatory (Infl) and granulating (Gran) phases. WF and THP-1 supernatants were analyzed for nine matrix metalloproteinases (MMP) and four tissue inhibitors of metalloproteinases (TIMP) by multiplex immunoassays. Our results demonstrated that: 1) WF from Infl VLU contained significantly increased concentrations of MMP-2, MMP-9, MMP-12, TIMP-1, and TIMP-2 compared to Gran WF; 2) WF from Gran VLU showed significantly increased levels of MMP-1, MMP-7, MMP-13, and TIMP-4 compared to Infl WF; 3) LPS- and WF-stimulation of THP-1 cells significantly increased the expression of several MMP compared to untreated cells; 4) Sulodexide treatment of both LPS- and WF-stimulated THP-1 significantly down-regulated the release of several MMPs. Our study provides evidence-based medicine during treatment of patients with VLU. WF from Infl and Gran VLU have different MMP and TIMP signatures, consistent with their clinical state. The modulation of proteolytic pathways in wound microenvironment by glycosaminoglycan sulodexide, provide insights for translating research into clinical practice during VLU therapy.

Chronic venous disease - Part I: Inflammatory biomarkers in wound healing.

Venous leg ulcers (VLUs) produce wound fluid (WF), as a result of inflammatory processes within the wound. It is unclear if WF from different healing phases of VLU has a peculiar biochemical profile and how VLU microenvironment affects the wound healing mechanisms. This study was conducted to evaluate the cytokine/chemokine profiles in WF from distinct VLU phases, in WF- and LPS-stimulated monocytes and treated with glycosaminoglycan Sulodexide, a therapeutic option for VLU healing. WF and plasma were collected from patients with VLU during active inflammatory (Infl) and granulating (Gran) phases. Demographics, clinical characteristics and pain measurements were evaluated. WF, plasma, and THP-1 supernatants were analyzed for 27 inflammatory mediators by multiplex immunoassay. Our results demonstrated that: 1) pain was significantly increased in patients with Infl compared to Gran VLU; 2) cytokine profile of Infl WF was found to be statistically different from that Gran WF, as well significantly increased respect to plasma; 3) LPS- and WF-stimulation of THP-1 cells significantly increased the expression of several cytokines compared to untreated cells; 4) Sulodexide treatment of both LPS- and WF-stimulated THP-1 monocytes was able to significantly down-regulate the release of peculiar inflammatory mediators. Our study highlighted the importance to understand biomolecular processes underlying CVI when providing treatment for chronic VLU. Identification of inflammatory biomarkers in leg ulcer microenvironment, may provide useful tools for predicting healing outcome and developing targeted therapies.

Pharmacologic treatment to improve venous leg ulcer healing

Pharmacologic treatment for venous leg ulcers (VLUs) is an adjuvant treatment to compression therapy. It encompasses a variety of plant-derived and synthetic compounds with properties that alter venous microcirculation, endothelial function, and leukocyte activity to promote VLU healing. These compounds are often referred to as venotonics or venoactive drugs but have also been referred to as edema-protective agents, phlebotonics, vasoprotectors, phlebotropics, and venotropics. The exact mechanism of their ability to heal VLUs is not known; however, clinical trials support their efficacy. This evidence-based review assesses randomized clinical trials and meta-analyses with the objective of determining the effectiveness of venotonics to promote VLU healing.

Glutathione Depletion Is Linked with Th2 Polarization in Mice with a Retrovirus-Induced Immunodeficiency Syndrome, Murine AIDS: Role of Proglutathione Molecules as Immunotherapeutics.

ABSTRACT Injection of the LP-BM5 murine leukemia virus into mice causes murine AIDS, a disease characterized by many dysfunctions of immunocompetent cells. To establish whether the disease is characterized by glutathione imbalance, reduced glutathione (GSH) and cysteine were quantified in different organs. A marked redox imbalance, consisting of GSH and/or cysteine depletion, was found in the lymphoid organs, such as the spleen and lymph nodes. Moreover, a significant decrease in cysteine and GSH levels in the pancreas and brain, respectively, was measured at 5 weeks postinfection. The Th2 immune response was predominant at all times investigated, as revealed by the expression of Th1/Th2 cytokines. Furthermore, investigation of the activation status of peritoneal macrophages showed that the expression of genetic markers of alternative activation, namely, Fizz1, Ym1, and Arginase1, was induced. Conversely, expression of inducible nitric oxide synthase, a marker of classical activation of macrophages, was detected only when Th1 cytokines were expressed at high levels. In vitro studies revealed that during the very early phases of infection, GSH depletion and the downregulation of interleukin-12 (IL-12) p40 mRNA were correlated with the dose of LP-BM5 used to infect the macrophages. Treatment of LP-BM5-infected mice with N-(N-acetyl-l-cysteinyl)-S-acetylcysteamine (I-152), an N-acetyl-cysteine supplier, restored GSH/cysteine levels in the organs, reduced the expression of alternatively activated macrophage markers, and increased the level of gamma interferon production, while it decreased the levels of Th2 cytokines, such as IL-4 and IL-5. Our findings thus establish a link between GSH deficiency and Th1/Th2 disequilibrium in LP-BM5 infection and indicate that I-152 can be used to restore the GSH level and a balanced Th1/Th2 response in infected mice. IMPORTANCE The first report of an association between Th2 polarization and alteration of the redox state in LP-BM5 infection is presented. Moreover, it provides evidence that LP-BM5 infection causes a decrease in the thiol content of peritoneal macrophages, which can influence IL-12 production. The restoration of GSH levels by GSH-replenishing molecules can represent a new therapeutic avenue to fight this retroviral infection, as it reestablishes the Th1/Th2 balance. Immunotherapy based on the use of pro-GSH molecules would permit LP-BM5 infection and probably all those viral infections characterized by GSH deficiency and a Th1/Th2 imbalance to be more effectively combated.

Characterization of oversulfated chondroitin sulfate rich in 4,6-O-disulfated disaccharides in breast cyst fluids collected from human breast gross cysts.

Glycosaminoglycans (GAGs) from breast cyst fluid (BCF) of gross cysts, subdivided into apocrine and flattened, directly collected from 27 gross‐cystic‐breast‐disease (GCBD)‐affected women were analysed. Heparan sulfate, not further investigated, and chondroitin sulfate were identified. This last polysaccharide, in a content of 25–27 µg ml−1 BCF and having a high molecular mass (~20 000–22 000), was found rich in glucuronic acid (~96%–98%) and mainly sulfated in position 4 of the N‐acetyl‐galactosamine (~60%–64%). Moreover, the presence of ~19%–24% of uncommon 4,6‐O‐disulfated disaccharides CS‐E inside the polysaccharide chains with a high charge density of ~1.15–1.20 was determined. No substantial differences between apocrine and flattened cysts were observed. The current study describes the first effort to examine the yield and distribution of complex macromolecules like GAGs in BCF, and the understanding of their structure may help explain some functions associated with physiological and pathological conditions. Copyright