Characteristics of Venous Thrombi in Animal Models of Obesity, Diabetes and Hypertension
Authors: Santiago-Bassora Fernanda Dutra , Barnabé Aline , Zapponi Kiara Cristina Senger , Romano Anna Virginia Calazans , Gonçalves Any Elisa de Souza Smidt , Saad Mario Jose Abdalla , Diaz Jose Antonio , De Paula Erich Vinicius , Annichino-Bizzacchi Joyce Maria 
Published: October 2019

Abstract
Introduction: Deep venous thrombosis (DVT) is defined as a vessel occlusion of the venous system and the pathophysiology is based on the change in blood flow, the vascular wall and/or blood elements. Obesity, diabetes and hypertension are being investigated as risk factors for DVT, since some clinical studies have suggested a correlation between DVT and arterial disease.
Objective: In this study we evaluated the size and components of venous thrombus in animals with risk factors for arterial disease.
Methods: The study was approved by the ethics committee on animal use of the Unicamp. Forty-five male Wistar and 15 spontaneous hypertensive rats (SHR) were separated into 4 groups of 15 animals. The control group (CTL) consisted of Wistar rats fed with standard chow diet; the obese group consisted of Wistar rats fed with a hyperlipidemic diet for 8 weeks; the diabetic group consisted of Wistar rats with diabetes induced by streptozotocin and were fed with chow diet; and the hypertension group consisted of SHR rats fed chow diet. All groups had DVT-induced stasis at 12 weeks old. After 3 hours, each animal was sacrified and the thrombus was removed. The weight of the thrombus was evaluated. Tissue factor (TF), and the presence of macrophages (M1 and M2) and endothelial cells were evaluated in thrombi using immunohistochemistry (IHC) and Western Blot technique.
Results: The phenotype of obesity characterized by an increase in body weight was confirmed only in the group fed with hyperlipidemic diet (CTL = 420 g ± 46 g vs Obese = 557 g ± 62 g vs Diabetes = 399 ± 104 vs Hypertension 259 ± 21; p ≤0.001) An increase in epididymal fat was observed only in the obese group (CTL = 6.5 ± 2.3 vs Obese = 10 ± 3.3 vs Diabetes = 4.9 ± 2.4 vs Hypertension 2.6 ± 0.3; p ≤0.001). Increased blood fast glucose was observed in diabetic group (CTL = 107 ± 19 vs Obese = 147 ± 12 vs Diabetes = 442 ± 91 vs Hypertension 149 ± 30; p ≤0.001). There was no difference between thrombus weight between control and experimental groups (CTL = 0.06 ± 0.01 vs Obese = 0.024 ± 0.08 vs Diabetes = 0.0073 ± 0.01 vs Hypertension = 0.031 ± 0.02; p =0.228). No differences were observed in the presence of macrophages, and endothelial cells were not detected in our samples. Western blot showed lower TF expression in all experimental groups (obese, diabetes and hypertension) compared to controls (CTL = 31 ± 2 vs Obese = 14 ± 0.4 vs Diabetes = 2.1 ± 0.9 vs Hypertension = 2.5 ± 1; p =0,002), which was consistent with the qualitative IHC analysis. No additional qualitative differences were observed by IHC regarding P-selectin, macrophages and endothelial cells.
Conclusion: Obesity, diabetes and hypertension have been recognized as risk factors for DVT. However, no differences could be observed in thrombus weight and in the presence of endothelial cells and macrophages within the thrombus in the well-established venous stasis rat model in young rats at this time point. The lower expression of TF observed in our study could be due to differences in the composition of other cellular components of the thrombi, or in the source of TF in these different models. Additional studies are warranted to investigate these hypotheses at other time points and older rats.

Disclosures
No relevant conflicts of interest to declare.