Circulating Endothelial Cells Are Increased in Post-Thrombotic Syndrome Patients

BACKGROUND: Post-thrombotic syndrome (PTS) is a common complication presented by patients with lower limbs deep vein thrombosis (DVT) that is characterized by pain, edema, cramps, itching, paresthesia, hyperpigmentation, and/or wound. PTS results in significant disability, and patients with severe degrees have a quality of life comparable to patients with cancer. From 20-50% of proximal DVT patients develop PTS despite optimal anticoagulant therapy, and between 5 to 10% of them present severe PTS. The diagnosis is based on clinical signs and several clinical classifications have been applied to graduate its severity, whereas the ISTH recommends the use of Villalta scale. PTS physiopathology remains unclear, but the inflammatory response to acute thrombosis, as well as to the fibrosis of venous valves and walls, seem to play an important role. Circulating endothelial cells (CEC) are infrequently detected cells derived from vascular wall or recruited from bone marrow. They have been related to hemostasis, platelet and leukocytes interaction to the vessel wall, and endothelial injury. Increased numbers of CEC are observed in ischemia, vascular trauma and have been associated to angiogenic potential. The aim of this study was to analyze CEC numbers in patients with a history of previous DVT and PTS, comparing them with DVT patients without PTS and healthy controls. We also aimed to compare CEC levels among patients with different degrees of PTS severity. Plasma D-dimer and serum IL8 levels were also determined, respectively by turbidimetric and nephelometric methods.

PATIENTS AND METHODS: CEC percentage were determined in PTS patients (N=19), which were subdivided according to their score on Villalta scale; DVT patients with no post-thrombotic syndrome (N=9) and healthy controls (N=19). PTS patients and controls were matched by gender, ethnic origin and age ±5 years, and were also included. Blood (20mL) were collected in EDTA vacuum tubes, and 100μL of whole blood (with leukocyte counting between 5-10x10³/μL) were used on flow cytometry analyzes. Different antibodies were included in order to better detect CEC, and two different panels were applied (panel 1: CD45-, CD144+, CD31+, CD133-; panel 2: CD45-, anti-VEGFR2+, CD31+, CD133-).

RESULTS: Patients with PTS presented higher percentage of CEC, comparing to the other groups. Mild PTS patients presented higher levels of CEC using both panels in comparison to patients without PTS (P= 0.015, P= 0.019 respectively) as well as to CTR (P= 0.077, P= 0.040 respectively). The moderate+severe PTS patients presented higher levels of CEC using panel 2 both in comparison to patients without PTS (P= 0.008) and CTR (P= 0.013). CD144+ cells were slightly increased in moderate+severe PTS patients comparing to patients without PTS (P= 0.054). Both IL8 (med= 21.85 vs med=13.84 pg/mL; P=0.011) and D-dimer (med= 0.475 vs med= 0.29mg/L; P=0.044) were increased in PTS patients in comparison to patients without PTS, respectively. As CEC could eventually be derived from the residual venous thrombosis (RVT), we also dichotomized patients between those with and without RVT. The levels of CEC were similar between patients with or without RVT.

CONCLUSIONS: Our results suggested that PTS may be associated with higher CEC levels. It is believed that CEC has angiogenic properties and that they may be increased on oxidative stress situations. In this context, our findings may suggest that CEC takes part on vascular-mediated PTS pathophysiology. Also the higher levels of D-dimer and IL8 may indicate that PTS patients were experiencing a pro-inflammatory condition.