Antithrombotic effect of grape seed proanthocyanidins extract in a rat model of deep vein thrombosis Yunjian Zhang, PhD, MD, Hanping Shi, PhD, MD, Wenjian.

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Presentation transcript:

Antithrombotic effect of grape seed proanthocyanidins extract in a rat model of deep vein thrombosis Yunjian Zhang, PhD, MD, Hanping Shi, PhD, MD, Wenjian Wang, PhD, MD, Zunfu Ke, PhD, MD, Ping Xu, MD, Zhiqiang Zhong, PhD, Xiaoxi Li, PhD, MD, Shenming Wang, PhD, MD, FACS Journal of Vascular Surgery Volume 53, Issue 3, Pages 743-753 (March 2011) DOI: 10.1016/j.jvs.2010.09.017 Copyright © 2011 Terms and Conditions

Fig 1 Grape seed proanthocyanidins extract (GSPE) reduced thrombosis formation. The thrombus (a) length and (b) size increased and reached peak values at about days 5 to 7. The longitudinal axis length and weight of thrombus in the GSPE group were significantly smaller than the control (since day 3); (c) GSPE significantly reduced thrombus size (weight in g/length in cm). **P < .01. Journal of Vascular Surgery 2011 53, 743-753DOI: (10.1016/j.jvs.2010.09.017) Copyright © 2011 Terms and Conditions

Fig 2 The thrombus weight has a linear correlation with the cytokine production of (a) interleukin (IL)-6 (r = 0.874), (b) IL-8 (r = 0.878); and (c) tumor necrosis factor (TNF)-α (r = 0.919), but this was not shown in the grape seed proanthocyanidins extract (GSPE) group (IL-6, r = 0.005; IL-8, r = 0.029; TNF-α, r = 0.353). Journal of Vascular Surgery 2011 53, 743-753DOI: (10.1016/j.jvs.2010.09.017) Copyright © 2011 Terms and Conditions

Fig 3 Mature thrombi (TH) were detected by hematoxylin and eosin staining in paraffin sections of inferior vena cava (IVC) biopsy specimens at day 3 after IVC ligation. Thrombi compression and adhesion, fibrin strands, and platelet aggregates were observed to attach to a grossly intact vein wall (a, b). However, these were not found in the grape seed proanthocyanidins extract (GSPE) (c) and sham group (d) (original magnification ×200). Journal of Vascular Surgery 2011 53, 743-753DOI: (10.1016/j.jvs.2010.09.017) Copyright © 2011 Terms and Conditions

Fig 4 Analysis of specimens by transmission electron microscope: (a) prethrombotic vein wall endothelium (original magnification ×6600); (b) after inferior vena cava ligation, inflammation cell migration, adhesion, and infiltration at day 1 (original magnification ×5200); (c) endothelial cell necrosis at day 3 (original magnification ×11500); (d) subendothelial extracellular matrix exposure and contact with blood cell (original magnification ×8900); (e) the integrity of endothelium was destroyed in the control group at day 5 (original magnification ×3900); (f) thrombogenesis at day 1 (original magnification ×5200); (g) an activated mononuclear macrophage releases cellular adhesion molecules to induce the cell adhesion (original magnification ×5200) on day 3; (h) grape seed proanthocyanidins extract maintained the integrity of endothelium at day 5, and the cell-cell junction closed without leukocyte adhesion and infiltration (original magnification ×5200). EC, Endothelial cell; EC(n), necrotic EC; M, mononuclear macrophages/monocytes; RBC, red blood cell; Th, thrombi; vw, vein wall. Journal of Vascular Surgery 2011 53, 743-753DOI: (10.1016/j.jvs.2010.09.017) Copyright © 2011 Terms and Conditions

Fig 5 CD34 and vascular endothelial growth factor (VEGFR)-2 expression in the endothelium. CD34 (a, b) and VEGFR2 (g, h) staining in the endothelium of sham group are shown as above. Representative photomicrographs of immunohistochemistry-stained paraffin sections of vein biopsy specimens are shown. c, d, The CD34 expression of endothelium in control group at day 3 shows negative or weak staining in the endothelium in the control, representing an endothelium loss (original magnification ×200).e, f, Endothelial cells were positively stained in the grape seed proanthocyanidins extract (GSPE) group at day 3, indicating the maintenance of the endothelium (original magnification ×200). i, j, VEGFR-2 expression in the control specimens was negatively stained on the major part of the endothelium but weakly positive at the site of the endothelium without thrombus or leukocyte adhesion on day 3 (original magnification ×200). k, l, The positively-stained areas were detected at the endothelium in GSPE group specimens at day 3 (original magnification ×200). m, The bar chart representing the percentage of CD34 stained at the endothelium shows that GSPE significantly increased CD34 expression compared with control (P < .05). n, The two bars representing the expression of VEGFR-2 in control and GSPE treatment show that the VEGFR2 expression in the GSPE treatment group is significantly higher than that of control, calculated by the staining area/total area of the inferior vena cava endothelium (P < .05). The solid line arrows point to the positive staining area; the dotted line arrows point to the negative staining area. TH,, thrombus; vw, vein wall. *P < .05. 100×: a, c, e, g, i, k;/200×: b, d, f, h, j, l. Journal of Vascular Surgery 2011 53, 743-753DOI: (10.1016/j.jvs.2010.09.017) Copyright © 2011 Terms and Conditions

Fig 6 Representative photomicrographs of immunohistochemistry-stained paraffin sections of vein biopsy specimens show P-selectin expression in the endothelium and platelets. Expression of P-selectin in (a, b) the control group was higher than in the (c, d) grape seed proanthocyanidins extract (GSPE) group at day 3, particularly in the area of thrombus and endothelial cells. At the site of high P-selectin expression, leukocyte recruitment can be seen. e, P-selectin expression is shown in the sham group. f, The bar chart shows that GSPE decreased P-selectin expression, calculated by the staining area/total area of the IVC endothelium (P < .05). Arrows point to the endothelium; triangles point to the strongly positive luminal area. TH, Thrombus; vw, vein wall. *P < .05. 100×: a, c, e;/200×: b, d. Journal of Vascular Surgery 2011 53, 743-753DOI: (10.1016/j.jvs.2010.09.017) Copyright © 2011 Terms and Conditions

Fig 7 Expression is shown of von Willebrandt factor (vWF) and ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) in cryostat sections of (a, b, c) prethrombosis, (d, e, f) control group, and (g, h, i) grape seed proanthocyanidins extract (GSPE) group. b, vWF expression of prethrombotic vein endothelium was weakly stained. After inferior vena cava ligation, vWF expression in the control group was upregulated at day 1. e, Endothelium and thrombus were both strongly positive stained. h, vWF expression of in the GSPE group was weaker than the control. On the contrary, the expression of ADAMTS13 in the (f) control group was weaker than in the (i) GSPE group. Black arrows point to the endothelium shown in the hematoxylin and eosin (HE)-stained slice. White arrows point to the prethrombotic endothelium. Blue arrows point to the postthrombosis endothelium in control group. Pink arrows point to the postthrombotic endothelium in the GSPE group. Th, Thrombus; vw, vein wall. 100×: a, b, c, e, g, h, i;/200×: d, f. Journal of Vascular Surgery 2011 53, 743-753DOI: (10.1016/j.jvs.2010.09.017) Copyright © 2011 Terms and Conditions

Fig 8 Vascular cell adhesion molecule (VCAM)-1 and intracellular cell adhesion molecule (ICAM)-1 expression is shown in thrombi harvested at days 1, 3, 5, and 7 and lysed with the lysis buffer. A 15-μg protein lysate was subjected to electrophoresis on 8% sodium dodecyl sulfate polyacrylamide gels to detect VCAM-1 and ICAM-1 with VCAM-1 antibody (1:500) and ICAM-1 antibody (1:500), respectively. VCAM-1 and ICAM-1 expression in the grape seed proanthocyanidins extract (GSPE) group were significantly lower than that of the control. **P < .01. Journal of Vascular Surgery 2011 53, 743-753DOI: (10.1016/j.jvs.2010.09.017) Copyright © 2011 Terms and Conditions