Venous lower limb microenvironment: an open window on the Chronic Venous Disease

Chronic Venous Disease (CVeD) is a common pathologic condition highly spread in the Western country population generally consisting in a series of different chronic venous disorders affecting lower limbs (Wound Repair Regen. 2009; 17(6):763-71; Bergan et al, N Engl J Med. 2006; 355(5):488-98). One of the well-known hallmarks of the CVeD is a gradual transition from the appearance of the first morphological and functional aberrations (e.g. telangiectasia, reticular and varicose veins) corresponding to C0-C2 classes of the Clinical, Etiological, Anatomical, and Pathophysiological (CEAP) classification of CVeD to substantial impairments of the venous system, as edema, eczema, lipodermatosclerosis and healed or active ulcers, representing the C3-C6 classes of the CEAP (J Vasc Surg. 2004; 40(6):1248-52). Different risk factors, including the aging population, familiar history, obesity, diabetes, female sex, pregnancy, previous leg injury or venous thrombosis, venous insufficiency, often promote the occurrence of this pathological state with a subsequent sharp decrease of patient quality of life and a progressive increase of morbidity and mortality (Flore et al, Eur Rev Med Pharmacol Sci. 2015; 19(4):557-62). As previously mentioned, Venous Leg Ulcer (VLU) is usually considered the final stage of CVeD and consists in a real skin ulceration of the lower limbs which may become chronic (CVLU) showing slow tendency to heal associated with pain, swelling, edema and different skin changes, often as result of Chronic Venous Insufficiency (CVI). (Mannello et al, Expert Rev Mol Diagn. 2014; (6):737-62; Martin et al, Br J Dermatol. 2015; 173(2):370-8). At the onset of this pathological state can be identified several biochemical, physical and hemodynamic alterations promoting microcirculation disturbances, endothelium activation and leucocytes recruitment with a subsequent development of hypertension and inflammatory reaction. (Raffetto et al, Thromb Res. 2009; 123: S66-71; Takase et al, J Vasc Surg 2004;39:1329- 34). All these events pave the way to an intense oxidative stress and a plentiful release of cytokines, chemokines, grow factors and proteolytic enzymes, as the Matrix Metalloproteinases (MMP), characterizing the ulcer microenvironment (Ligi et al, Biochim Biophys Acta. 2016; 1862(10):1964-74; Pocock et al, Vascular Cell. 2014, 6:24). The MMPs are a Zn2+-dependent endopeptidase family differentially involved in the four sequential phases of the physiological wound healing process (homeostasis, inflammation, granulation and remodeling), other than in the whole CVeD (Ligi et al, Biochim Biophys Acta. 2016; 1862(10):1900-8; Reinke, Sorg Eur Surg Res. 2012; 49:35–43). These proteolytic enzymes with other biochemical factors, including the Transforming Grow Factor beta (TGF-β) superfamily and the soluble endoglin (sEng), could be strictly involved in an aberrant Extracellular Matrix (ECM) remodeling and in the arrest of wound healing in the inflammatory phase (Mannello et al, Int J Mol Sci. 2017; 18(10): 2206; Barrientos et al, Wound Rep Reg. 2008;16: 585–601). Thus, according with the main aim of this three-year research project to investigate the lower limb microenvironment in different CVeD stages, the first step was to determine the expression and modulation of these mediators as some possible biological hallmarks responsible of the ulcer chronicity and the appearance of persistent skin damages. In particular, we have determined the concentration of MMPs, Tissue Inhibitors of Matrix Metalloproteinases (TIMP), cytokines, all three TGF-β isoforms and sEng and their possible interactions in non-healing and healing wound fluid samples. We have also investigated the release of MMPs, TGF-β isoforms and sEng in supernatants of THP-1 monocyte cell line exposed to various stimuli. These analyses are realized through multiplex suspension immunomagnetic assays. In addition, polyacrylamide-gelatin zymography is carried out on THP-1 supernatant samples to evaluate the activity of the gelatinases MMP-2 and MMP-9 strongly involved in the healing process. The collected data analysis has shown that the TGF-β3 and endoglin concentration appeared significantly increased respectively in non-healing and healing ulcer wound fluid. This evidence confirms both the well-known role of TGF-β3 to counteract the TGF-β1 effects enhancing the release of inflammatory mediators contributing to the ulcer chronicity (Pakyari et al, Adv Wound Care (New Rochelle). 2013; 2(5):215-224) and the sEng involvement in the decrease of inflammation promoting the wound progression in the healing process (Barbara et al, J Biol Chem. 1999; 274(2):584-94). Interesting modulation of the gelatinases, TGF-β isoforms and sEng is also picked-out by the in vitro quantitative and qualitative results. Moreover, statistical correlation among MMPs, TIMPs, cytokines, TGF-β isoforms and endoglin are preliminary performed resulting significant in some cases. All these evidences have allowed us to observe that the modulation of the expression of TGF-β isoforms and its accessory receptor endoglin are probably a faithful mirror of their distinct roles in the healing process. The second step of our work was to investigate the modulation of MMP expression and activity after two clinical treatments: the gold-standard compression therapy and the most recent sclerotherapy (Rabe et al, Phlebology. 2014; 29(6):338-54). The first therapeutic option is aimed to re-establish a regular blood flow removing the venous reflux and promoting the physiological drainage through the wearing of garments or bandages with different compression degree, while the second induces a chemical ablation of the treated vein through the direct injection of liquid or foamed sclerosing detergents (polidocanol and sodium tetradecylsulfate). In line with the aim of the study to characterize the venous microenvironment retracing the CVeD stages, the quantitative determination of MMPs is performed in plasma samples of healthy volunteers and C4a/C4b patients underwent to three different conditions: standing and lying position, stocking compression. In particular, these patients are affected by lipodermatosclerosis and hyperpigmentation characterized by venous hypertension, endothelial disfunction, abnormal fibrinolysis, fibrosis and matrix degradation accompanied by skin hardening and discoloration. The analyses are realized through multiplex suspension immunomagnetic assays. Electrophoretic assays are performed to evaluate the expression of MMPs and some serum/plasma proteins (fibrinogen, D-dimer, albumin, globulins and lipoproteins) in blood samples following the treatment with increasing concentrations of the two sclerosant agents. In addition, zymographies are carried out on the plasma samples treated in the same way to determine eventual MMP modulation activity. In vitro test was carried out to observe the lytic effects of sclerosing detergents on HECV human endothelial cells. The collected data showed that the MMP expression in C4a/C4b patients is more susceptible to the limb positions and compression treatment than volunteers suggesting a potential modulation of the proteolytic microenvironment deeply involved in the hemodynamic and tissue alterations characterizing this pathologic state (Raffetto et al, Thromb Res. 2009; 123: S66- 71). Interestingly, the in vitro assay allowed us to observe differences in MMP and plasma proteins expression probably due to interactions based on the different chemical nature of the sclerosant agents (Parsi, Phlebology. 2015;30(5):306–315). According to the literature, we have also detected a greater susceptibility of HDL (increase) and LDL (decrease) to the sclerosing treatment than the other plasma and serum proteins considered in our study (Pownall et al, Biochemistry. 2006; 45(38): 11514–11522). Moreover, the in vivo assays on the HECV revealed evident morphological changes induced by the treatment with increasing detergent concentrations confirming an increased cellular permeability probably accompanied by cellular activation and different cellullar death mechanisms. According with the guide line of the whole study, the last step of the project is focused on clarifying the modulation of MMP activity and some biochemical mediator expression strictly involved in the initial stages of CVeD corresponding to telangiectasias, reticular and varicose veins (C1/C2). These pathological states are equally characterized by a sequence of structural and cellular dysfunctions, extracellular matrix degradation and inflammation (Pascarella et al, Semin Vasc Surg. 2005;18(1):2-4). In particular, zymography and multiplex suspension immunomagnetic assays are performed in C1/C2 plasma samples to determine a possible modulation of MMP proteolytic activity and other biochemical mediators expression at different times (T0, T30’, T45’, T3h, T24h, T7d, T30d) following the treatment with low doses of the sclerosant detergent polidocanol. Our quantitative analysis showed a sequential increase of some growth factors, as bFGF, PDGFbb and VEGF well-known for their angiogenic and remodeling effects in vascular diseases, within 30 days from the treatment. In this regard, we have also observed a rapid release of some inflammatory cytokines, including IL-1β, IL-7, IL-8, IL-12, IL-13, IL-17, IFN-γ, TNF-α, immediately after the treatment reducing at long-term. All these results might confirm the literature evidences about the pro-angiogenic, pro-coagulant and pro-inflammatory effects of polidocanol at low concentration due to its possible interaction with numerous circulating cells, including leucocytes and platelets induced to release inflammatory mediators (Parsi et al, Eur J Vasc Endovasc Surg 34, 731-740,2007; Connor et al, Eur J Vasc Endovasc Surg (2015) 49, 426-431). Although a decrease in MMP-2 proteolytic activity is more appreciable than those of MMP- 9 especially up to the first hours after the treatment, the low concentrations of the sclerosant seem broadly to induce slight effects on these MMPs. This evidence might be consistent with a proteolytic activity modulation also based on the chemical nature of the detergent. Furthermore, it should not be forgotten that the low doses of polidocanol considered in this study cause only mild damages on the vessel endothelial lining as well as on other tissue and circulating cells stimulating their release of different inflammatory mediators and growth factors able to promote the MMP expression and activity (Sprague et al, Biochem Pharmacol. 2009; 78(6): 539–552). Thus, according with our study results we might conclude that MMPs are a common factor of the different CVeD stages starting from the appearance of the first venous functional abnormalities to significant tissue damages which may often evolve in invalidating conditions. An interesting implication of this evidence consists in setting MMPs in the biochemical framework characterizing venous disorders in which take place mutual interactions able to impair physiologic balances and compromise the biologic machine of the venous microenvironment. In this regard, it might be useful to deepen the mechanisms which are triggered by both the involved mediators and the pharmacologic treatments to prevent irreversible impairments and select the most suitable approach.