ANTI-VASCULAR ENDOTHELIAL GROWTH FACTOR THERAPY ATTENUATES ...

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  • Vascular endothelial growth factor (VEGF) is a pro-inflammatory cytokine implicated in increasing endothelial cell permeability, inducing the expression of endothelial adhesion molecules, and acting as a monocyte chemoattractant in allograft rejection. Up-regulation of VEGF is associated with hepatic I/R injury and anti-VEGF treatment attenuates hepatic I/R injury.
  • The aim of this study was to explore the cytoprotective mechanisms of anti-VEGF therapy in a model of mouse hepatic warm I/R injury.
  • As shown in this slide, mouse partial hepatic warm ischemia was induced by clamping the left & middle hepatic lobes for 90 min. Mice were sacrificed at 0-6 hours of reperfusion, and liver samples were collected. In the first set of experiments, VEGF gene and protein expression were examined.
  • In the second set of experiments, the effects of VEGF blockade after treatment with anti-VEGF Ab were assessed at 6 hours post reperfusion. Experimental animals were divided into 3 groups Sham group, without treatment and without clamping the lobes. Control group, mice were treated with control Ab at day -1 and just before the ischemia. Anti-VEGF group, mice were treated with anti-VEGF polyclonal Ab.
  • This slide shows time course of VEGF gene and protein expression in hepatic I/R. Ninety minutes of ischemia alone triggered a significant increase of VEGF mRNA expression, compared with naïve controls. This expression increased further in the post-ischemia period, peaked at 2 hours of reperfusion, and decreased thereafter. VEGF protein levels increased progressively during I/R and peaked at 6 hours of reperfusion.
  • Next, we assessed whether the blockade of VEGF protected mouse livers against hepatic I/R injury. Anti-VEGF treatment significantly reduced serum GPT levels after 6 h of reperfusion, as compared with controls.
  • This slide shows histological findings after 6 h of reperfusion The histological severity of IRI was graded using Suzuki’s criteria. In this classification, sinusoidal congestion, hepatocyte necrosis, and ballooning degeneration are graded in order of increasing tissue injury on a scale ranging from 0 to 4. With a score of 7.87, the control group showed severe sinusoidal congestion and hepatocyte necrosis. In contrast, the anti-VEGF group had good preservation of lobular architecture without edema, congestion, or necrosis reflected by a Score of 1.0.
  • We measured MPO activity as an index of neutrophil infiltration in the liver. Anti-VEGF treatment showed significantly decreased MPO activity, compared with conrols.
  • We next examined whether anti-VEGF treatment affected the expression of pro-inflammaory cytokines, chemokines and adhesion molecules known to have an important function in propagating the pathogenesis of IRI. We performed RT-PCR for pro-inflammatory cytokines TNF-a and IFN-g known to be upregulated during hepatocellular injury. Anti-VEGF treatment significantly down-regulated the expression of these cytokines , compared with controls. The expression of E-selectin , (pause) an adhesion molecule involved in mediating initial leukocyte te th ering on the surface of endothelial cells was markedly suppressed in the anti-VEGF group. The expression of potent leukocyte chemoatractants IP-10 and MCP-1 was also significantly inhibited by using anti-VEGF therapy.
  • This slide shows Immunohistochemical staining for intrahepetic pan leukocytes, T cells and macrophages after reperfusion. In the control group we see increased expression of CD45, CD3, and Mac positive cells compared with the anti-VEGF group.
  • This slide shows TUNEL-assisted detection of apoptosis. Anti-VEGF treatment had significantly less TUNEL positive cells, compared with control mice.
  • We also evaluated anti-apoptotic, pro-apoptotic and HO-1protein expression. Anti-VEGF therapy enhanced anti-apoptotic Bcl-2, Bcl-xl, and HO-1protein expression, compared with controls. In contrast, the expression of pro-apoptotic Bax was suppressed by anti-VEGF treatment.
  • So we can conclude that Anti-VEGF treatment prevented hepatic warm I/R injury. Anti-VEGF treatment inhibited T-cell and monocyte/macrophage infiltration through the inhibition of cellular adhesion molecules, decreased the expression of pro-inflammatory cytokines/ckemokines. Anti-VEGF therapy also prevented apoptosis, up-regulated expression of anti-apoptotic (Bcl-2/Bcl-xl)/antioxidant (HO-1) protective molecules, and depressed pro-apoptotic (Bax) protein levels. By selectively modulating leukocyte trafficking patterns, VEGF plays a paramount role in the pathophysiology of hepatic I/R injury.
  • So we can conclude that Anti-VEGF treatment prevented hepatic warm I/R injury. Anti-VEGF treatment inhibited T-cell and monocyte/macrophage infiltration through the inhibition of cellular adhesion molecules, decreased the expression of pro-inflammatory cytokines/ckemokines. Anti-VEGF therapy also prevented apoptosis, up-regulated expression of anti-apoptotic (Bcl-2/Bcl-xl)/antioxidant (HO-1) protective molecules, and depressed pro-apoptotic (Bax) protein levels. By selectively modulating leukocyte trafficking patterns, VEGF plays a paramount role in the pathophysiology of hepatic I/R injury.
  • ANTI-VASCULAR ENDOTHELIAL GROWTH FACTOR THERAPY ATTENUATES ...

    1. 1. ANTI-VASCULAR ENDOTHELIAL GROWTH FACTOR THERAPY ATTENUATES HEPATIC ISCHEMIA AND REPERFUSION INJURY S. Tsuchihashi 1 , B. Ke 1 , F. Kaldas 1 , R.W. Busuttil 1 , D.M. Briscoe 2 , J.W. Kupiec-Weglinski 1 1 Dumont-UCLA Transplant Center, Division of Liver and Pancreas Transplantation, Department of Surgery 2 Division of Nephrology, Children’s Hospital, Harvard Medical School
    2. 2. BACKGROUND 2. Up-regulation of VEGF is associated with hepatic ischemia/reperfusion (I/R) injury. 3. Anti-VEGF treatment attenuates hepatic I/R injury. (Boros P, et al. Transplantation 2001;72: 805) (Ke B, et al. Transplantation 2005;79:1078) 1. Vascular endothelial growth factor (VEGF) is a pro-inflammatory cytokine implicated in increasing endothelial cell permeability, inducing the expression of endothelial adhesion molecules, and acting as a monocyte chemoattractant in allograft rejection. (Reinders MEJ, et al. J.Clin.Invest 2003;112:1655)
    3. 3. AIM To explore the cytoprotective mechanisms of anti-VEGF therapy in hepatic I/R injury
    4. 4. Animals: male C57BL/6 mice. 25~30 g Model: Partial (left & middle hepatic lobes) warm I/R injury Ischemic Time : 90 min Reperfusion Time : 0, 2, 4, and 6 h (n=6/time point) METHODS Ischemia (90 min) 0 h Reperfusion Evaluation: 2 h 4 h 6 h VEGF mRNA (RT-PCR) VEGF protein (Western blot)
    5. 5. METHODS Experimental groups: -24 h Ischemia (90 min) 0 Reperfusion (6 h) <ul><li>Sham group (n=5) </li></ul><ul><li>Control group (n=8) </li></ul><ul><li>Anti-VEGF group (n=9) </li></ul>Sacrifice Anti-VEGF or control serum (0.8 ml i.p.) <ul><li>sGPT </li></ul><ul><li>Histology </li></ul><ul><li>MPO activity (neutrophil infiltration) </li></ul><ul><li>PCR (TNF-  , IFN-  , E-selectin, IP-10, MCP-1) </li></ul><ul><li>Immunohistochemistry (CD45, CD3, Mac) </li></ul><ul><li>TUNEL staining </li></ul><ul><li>Western blot (Bcl-2, Bcl-xl, Bax, HO-1) </li></ul>Evaluation (6 h after reperfusion):
    6. 6. 42kDa 42kDa  -actin VEGF  -actin VEGF Naïve 0h 2h 4h 6h Naïve 0h 2h 4h 6h After reperfusion After reperfusion Western blot RT-PCR VEGF expression in hepatic I/R *P < 0.05 vs Naive #P < 0.05 vs 0h or 4h *P < 0.01 vs Naive #P < 0.01 vs 0h, 2h, 4h 0 0.2 0.4 0.6 0.8 1 Naive 0h 2h 4h 6h VEGF mRNA/  -actin After reperfusion * * * # VEGF protein/  -actin After reperfusion 0 0.2 0.4 0.6 0.8 1 1.2 Naive 0h 2h 4h 6h * * # *
    7. 7. sGPT *p<0.01 0 500 1000 1500 2000 2500 3000 3500 4000 4500 Sham Control Anti-VEGF s GPT (IU/L) *
    8. 8. Sham Control Anti-VEGF X 100 X 400 Histology Suzuki’s score = 7.97±1.08 1.00±0.87 P<0.01
    9. 9. MPO activity (Neutrophil infiltration) *p<0.01 0 1 2 3 4 5 6 7 8 9 10 Sham Control anti-VEGF Units /gm *
    10. 10. RT-PCR *p<0.01 **p<0.05 Pro-inflammatory cytokines Chemokines Adhesion molecule 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 TNF-  /  -actin Sham Control Anti-VEGF TNF-  * IFN-  /  -actin 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Sham Control Anti-VEGF IFN-  * 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Sham Control Anti-VEGF E-selectin/  -actin E-selectin * MCP-1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Sham Control anti-VEGF MCP-1/  -actin ** IP-10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Sham Control anti-VEGF IP-10/  -actin **
    11. 11. CD45 CD3 Sham Control Anti-VEGF Mac Immunohistochemical staining (Pan leukocyte) (T cell) (Macrophage)
    12. 12. TUNEL Sham Control Anti-VEGF *p<0.01 0 5 10 15 20 25 30 Sham Control anti-VEGF TUNEL positive cells/field *
    13. 13. 42kDa 32kDa 28kDa 32kDa 23kDa  -actin HO-1 Bcl-2 Bcl-xl Bax Sham Control Anti-VEGF Western blot
    14. 14. <ul><li>prevented hepatic warm I/R injury. </li></ul><ul><li>inhibited leukocyte (neutrophil, T-cell, and monocyte/macrophage) infiltration through the inhibition of cellular adhesion molecule, decreased the expression of pro-inflammatory cytokines/chemokines. </li></ul><ul><li>prevented apoptosis, up-regulated expression of anti-apoptotic (Bcl-2/Bcl-xl)/antioxidant (HO-1) protective molecules, and depressed pro-apoptotic (Bax) proteins. </li></ul>SUMMARY Anti-VEGF treatment:
    15. 15. <ul><li>By selectively modulating leukocyte trafficking patterns, VEGF plays a paramount role in the pathophysiology of hepatic I/R injury. </li></ul>CONCLUSION

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