Sulodexide

Inflammation and endothelial dysfunction are important venous changes in patients with chronic venous disease (CVD). The use of the venoactive drugs remains an important treatment modality for patients with CVD, reducing the severity of the CVD-related symptoms and swelling but also reducing inflammation and protecting endothelial cells. In this research, the effects of the serum obtained from patients with CVD before and after sulodexide treatment were evaluated for in vivo and in vitro inflammatory markers and endothelial cell function.

Inflammatory markers (IL-6, matrix metalloproteinase-9 [MMP-9], vascular cell adhesion molecule-1 [VCAM-1], and von Willebrand factor [vWF]) from the incompetent great saphenous veins (GSVs) and from the systemic venous circulation were studied in 10 patients with CVD (C2s) before and after 2 months of sulodexide (2 × 500 lipasemic units/d) therapy. Serum obtained from the vein blood before and after sulodexide treatment was evaluated for in vitro cultured human umbilical vein endothelial cell function.

The serum collected from lower leg incompetent GSVs had significantly elevated levels of VCAM-1 (+29%, P < .001) compared with the serum from the systemic circulation. Endothelial cells exposed to the serum from the incompetent lower leg veins of the untreated CVD patients demonstrated higher stimulated synthesis of MMP-9 (+17%, P < .01), as well as increased markers of senescence (prolongation of population doubling time, β-galactosidase activity, and expression of p21 and p53 genes). CVD serum-induced senescent endothelial cells had a higher expression of genes regulating IL-6, MMP-9, VCAM-1, and vWF synthesis. The overall proinflammatory effect on endothelial cells by the serum collected from the incompetent GSVs was stronger as compared with the serum from the systemic circulation. Serum collected from the veins after sulodexide treatment caused lower levels of endothelial cell inflammatory markers as well as respective gene expression than serum obtained at the beginning of the study (before sulodexide treatment). Sulodexide application also reduced the inflammatory secretory activity of the senescent endothelial cells. Sulodexide treatment resulted in the decrease of the majority of the studied inflammatory parameters in both lower limb incompetent vein and systemic blood.

In patients with CVD, there are significant differences between circulating inflammatory markers analyzed from the lower leg incompetent GSV segments compared with the systemic circulation, indicating a higher inflammatory condition in CVD. Treatment with sulodexide reduces the proinflammatory and endothelial cell activation properties of the serum from patients with CVD.

The study documented the significant proinflammatory human vascular endothelial cell activation when exposed to the serum collected from the varicose veins as compared with the serum from the systemic circulation in patients with chronic venous disease (CVD). The inflammatory marker expression, endothelial dysfunction, and endothelial cell senescence transformation can be successfully controlled and downregulated by patients' exposure to the glycosaminoglycan (sulodexide) treatment. Further studies are needed to confirm if glycosaminoglycan application can prevent further CVD clinical progression due to potential CVD-related pathological processes' modulation and their downregulation.

The lipid peroxidation product 4-hydroxynonenal (HNE) may be involved in vascular endothelial cell damage by induction of oxidative stress, apoptosis, and loss of redox homeostasis. There is evidence that stimulation of endothelial cells with 4-HNE induces the activation of the nuclear factor erythroid 2-related factor 2/Kelch-like ECH-associated protein 1 (Nrf2/Keap-1) pathway. Sestrin2 protein (SESN2) is one of the key regulators of Nrf2 and is involved in the cellular response to oxidative stress. However, the function of SESN2 in HNE-induced endothelial injury is not yet understood. Sulodexide (SDX) is a mixture of glycosaminoglycans used in clinical practice in the treatment of chronic venous and arterial diseases. While SDX has well-documented endothelial protective properties, little is known about its antioxidant effects. The aim of this study was to elucidate the molecular mechanisms activated by SDX in human umbilical endothelial cells (HUVECs) under HNE-induced oxidative stress. In this experimental model, we decided to evaluate the anti-apoptotic and antioxidant potential of SDX and its effect on the SESN2/Nrf2/GSH pathway. HUVECs were treated with 25 _M HNE or HNE combined with 0.5 LRU/mL SDX for 4 hours. Cell viability, apoptosis and intracellular reactive oxygen species (ROS) production were assessed by MTT assay and fluorescence microscopy. The expressions of Bax, cleaved caspase-3, Keap-1 and Nrf2 were determined by Western blot analysis. The intracellular concentrations of reduced glutathione (GSH) and oxidized glutathione (GSSG) were measured by colorimetric assay. SESN2, glutamate-cysteine ligase catalytic subunit (GCLc) and glutathione synthase (GSS) were assessed using ELISA. RT-qPCR was performed to detect Nrf2, GCLc and GSS mRNA levels. Transient Nrf2 silencing was obtained by short interfering RNA (siRNA). We have demonstrated that SDX can reduce the negative impact of HNE on HUVECs. SDX significantly protected HNE-treated HUVECs from apoptosis (p<0.001) and oxidative stress (p<0.001). SDX treatment significantly reduced Bax (p<0.05) and cleaved caspase-3 (p<0.01) expression. Co-administration of HNE and SDX increased GSH content (p<0.001) and GSH:GSSG ratio (p<0.001) as well as decreased SESN2 concentration (p<0.001) and Nrf2 (p<0.01), GCLc (p<0.05) and GSS (p<0.01) gene expression compared with the HNE group. Moreover, we revealed a negative correlation between SESN2 levels and GSH concentrations (p<0.001). Nrf2 silencing significantly decreased the effect of HNE and SDX on the induction of GCLc and GSS genes. SDX also significantly ameliorated the increase of nuclear Nrf2 in response to HNE (p<0.05). The results confirmed that SDX may protect against HNE-induced endothelial damage through its antioxidant effect and modulation of the SESN2/Nrf2/GSH signaling pathway.