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dcyphr | Chronic Venous Insufficiency: Transforming Growth Factor-β Isoforms and Soluble Endoglin Concentration in Different States of Wound Healing

Abstract

Venous leg ulcers (VLU) are a growing issue in healthcare. Transforming growth factor-beta (TGF-beta) and Endoglin (CD105) help to regulate wound healing and inflammation. CD105 can be cleaved off of its normal site on the cell membrane, called soluble CD105 (sCD105). Then the sCD105 can bind to and lower TGF-beta’s signal. This will increase angiogenesis. This study looks at the presence of TGF-beta and sCD105 in wound fluid. They tested the effect of sulodexide on healing VLU. The increase of TGF-beta 3 found shows that it has a negative effect on wound healing. High levels of sCD105 reduce inflammation by affecting how white blood cells move across vessel walls, called leukocyte adhesion and transmigration. This favors wound healing. Glycosaminoglycan sulodexide increases the amount of sCD105 released, so it may accelerate wound healing.


Introduction

Venous leg ulcer (VLU) is a complication of chronic venous disease (CVeD). The cause of VLU is not well understood. We need to find out how to heal wounds that are hard-to-heal. Wound healing is usually divided into four stages: hemostasis, inflammation, granulation, and remodeling. Ulcers or wounds that do not heal in 6 months are stuck in inflammatory conditions, so they cannot progress through the four stages. This study looks at the microenvironment of the would bed, specifically wound fluid (WF). Right now, there is no biomarker to tell is if a wound is in the process of healing, or if it is not progressing. This study looks at TGF-beta isoforms and sCD105 because of their role in vascular inflammation and wound healing.

TGF-beta 1 and TGF-beta 2 are well understood, and both respond to injury and help in wound healing. TGF-beta 3 is not well understood. CD105 is an auxiliary receptor to TGF-beta type II receptor. CD105 controls inflammation related leukocyte adhesion and transmigration. Matrix metalloproteinase (MMP)-14 cleaves CD105 and creates soluble CD105 (sCD105). sCD105 downregulates TGF-beta. Creating sCD105 may change the shape of the endothelial cells, which is seen in CVeD. CD105 has a high expression level on vessel wall cells and activated macrophages. CD105 is found highly upregulated during wound healing, but has not been studied in VLU wound fluid. 

This study looks at the three TGF-beta isoforms and sCD105 in healing and non healing VLU. This study also looks at the release of TGF-beta isoforms and sCD105 from wound fluid stimulated monocytes, with and without glycosaminoglycan sulodexide. Sulodexide had anti-inflammatory effects in CVeD.     

                                             

Methods

30 patients with VLU were chosen for the study. Human monocyte THP-1 cell line was used for the cell culture. Magnetic Multiplex Immunoassay was performed to determine the TGF-beta isoforms and sCD105 levels in the wound fluid.


Results

This study looked at 30 VLU patients. Based on how much the patient had healed, the tissues samples were divided into two groups: inflammatory (Infl) and granulating (Gran). The average timespan of the ulcer was 41.6 months. The average size was 10.7cm. The Infl patients had higher pain than the Gran patients. TGF-beta 1 and 2 were not at different levels for the Infl and Gran patients. But TGF-beta 3 was significantly increased in Infl patients compared to Gran patients. sCD105 was decreased in Infl compared to Gran patients, which tells us that sCD105 may change during the healing process. 

Human macrophage cells were treated with the inflammatory wound fluid and sulodexide. The sulodexide alone did not change the TGF-beta or sCD105 levels. But, sulodexide plus either Infl or Gran wound fluid increased the level of sCD105. This means that sulodexide interacting with biomolecules in wound fluid will lead to the shedding of sCD105.


Discussion

Specific cell types and extracellular matrix components have a strictly coordinated sequence of events to allow wound healing to occur. This study revealed that TGF-beta 3 is elevated in Infl wound fluid compared to Gran wound fluid. This suggests that TGF-beta 3 downregulates TGF-beta 1 and 2, and promotes the release of pro-inflammatory cytokines. TGF-beta 3 may be part of the cause of chronic VLU. This is supported because neutrophils are known to release TGF-beta 3 and neutrophils have been identified in the ulcer bed of patients with VLU. 

sCD105 competes with membrane bound CD105 for leukocyte integrin binding. sCD105 can outcompete membrane bound CD105, which reduces the leukocyte transmigration and therefore reduces inflammation. sCD105 will work against the membrane CD105, so it  decreases angiogenesis, capillary formation and sprouting but increases the permeability of vessels. Sulodexide increases the sCD105, which affects TGF-beta and leukocyte adhesion and transmigration. Although membrane bound CD105 plays an important role in angiogenesis, soluble CD105 may increase wound closure and therefore would be the first successful treatment in patients with a venous leg ulcer resulting from chronic venous disease.

*Eng stands for membrane bound CD105 and sEng stands for soluble CD105