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329: Simvastatin Treatment Attenuates Obliterative Airway Disease Development in Rat Tracheal Allograft Model
S180
Abstracts
Results: Histopathological analysis of renal tissues demonstrated frequent arterial hyalinosis, glomerusclerosis, almost complete glomerular collapse and prominence of visceral epithelial cells that contain blebs and vacuoles. We also observed tubular changes pertaining to renal toxicity such as tubular atrophy/vacuolization, epithelial blebs, and hyalinization. Additionally, increase in the number of intratubular proteinaceous casts, proteinaceous material swollen proximal tubular epithelial cells with increased intracytoplasmic protein droplets were seen in Sirolimus treated recipients. Intrarenal mRNA expression (Mean ⫾ SEM, expressed as relative fold expression) of CTGF (125 ⫾ 18), renin (175 ⫾ 45), p22phox (400 ⫾ 216) and TGF- (85 ⫾ 12) was observed in SRL treated rat cardiac transplant recipients. Conclusions: These studies provide evidence that even a short-term course of Sirolimus exposure leads to arterial and glomerular hyalinosis protein droplets indicative of proteinuria. These changes occur in concert with activation of the RAS, oxidative stress and tissue repair pathways. 329 Simvastatin Treatment Attenuates Obliterative Airway Disease Development in Rat Tracheal Allograft Model J.O. Ropponen1,2, J.M. Tikkanen1, M. Hollmen1, R. Krebs1, M.A. Keranen1, K.B. Lemstrom1,2 1University of Helsinki, Helsinki, Finland; 2University of Helsinki, Helsinki, Finland Purpose: Retrospective analyses have implied that statin therapy has a role in the prevention of bronchiolitis obliterans syndrome (BOS). We investigated the role of simvastatin in the development of obliterative airway disease (OAD) using a rat model of OAD. Methods and Materials: In fully-mismatched non-immunosuppressed rat tracheal allografts, we used simvastatin at different doses (0.1 – 20 mg/kg orally, daily) to assess its effect on OAD development. The effects of simvastatin treatment on alloimmune activation were measured using immunohistochemistry and PCR. We also administered L-NAME to inhibit the nitric oxide (NO) pathway. Rats were harvested 3, 10 or 30 days after transplantation. Results: In tracheal allografts epithelial injury was seen 3 days after transplantation. At day 10, regeneration of the epithelium was noticed in simvastatin therapy groups but not in control group. There was no difference in OAD development between the groups at this timepoint. At day 10 simvastatin also reduced inflammation in the allografts by suppressing neutrophil accumulation. At 30 days, simvastatin therapy reduced OAD development (p⬍ 0.05) in all other doses except the lowest dose (0.1 mg/kg). L-NAME-mediated NO synthase inhibition negated partly but not totally the beneficial effects of simvastatin at 10 day and 30 day time-points. Conclusions: Simvastatin treatment effectively inhibits OAD development. According to our data, the beneficial effect of simvastatin seems to be partly NO-dependent. Together with the clinical data available, these results suggest a possible role for statins in the prevention of BOS. We are working on further evaluation of the mechanism of simvastatin beneficial effects. 330 Effects of the JAK3-Inhibitor R348 on Different Cell Types Involved in Obliterative Airway Disease J.B. Velotta1, T. Deuse1, C. Peter2, E. Masuda3, V. Taylor3, G. Park3, D. Carroll3, R.C. Robbins1, S. Schrepfer1 2Rigel Inc, South San Francisco, CA Purpose: Janus-kinase-(JAK)3 is crucial for signal transduction in immune cells.
The Journal of Heart and Lung Transplantation February 2009
Methods and Materials: Trachea from BN-donors were transplanted heterotopically into the greater omentum of Lew-rats. Recipients were treated with either R348 (40 mg/kg/d), rapamycin (3mg/kg/d), or left untreated for 28 days. Trachea specimens were evaluated histologically and tracheal epithelial mucin gene 4 (Muc4) mRNA levels were determined between all groups using quantitative real time RT-PCR. Prevention of cellular rejection was investigated between all groups using enzymeELISPOT assays.To assess the role of R348 on smooth muscle cells, platelet derived growth factor (PDGF)stimulated human smooth muscle cells (hSMCs) were incubated with each respective drug (100nM) and a MTT proliferation assay was performed. Results: R348 40 mg/kg/d treated recipients and syngeneic recipients contained moderate and outstanding levels of PAS-positive goblet cells, respectively. Both rapamycin 3 mg/kg/d and untreated recipients expressed no PAS-positive goblet cells. These histological results were confirmed by qRT-PCR that showed R348 40 mg/kg/d treated recipients had 176-fold greater Muc4 RNA levels compared to untreated recipients (1.04 ⫾ 0.9 vs. 0.0059 ⫾ 0.003, p⬍0.02) and also significantly expressed 16-fold greater Muc4 RNA levels versus rapamycin 3 mg/kg/d treated recipients (1.04 ⫾ 0.9 vs. 0.06 ⫾ 0.1, p⬍0.02). R348 40 mg/kg/d treated recipients expressed similar Muc4 RNA levels compared to syngeneic recipients. Both, R348 and rapamycin, significantly reduced the Th1 (IFN- ␥) and Th2 (IL-4) immune response as evidenced by decreased spot frequencies of IFN-␥ (1064 ⫾ 117 vs. 366 ⫾ 50 and 194 ⫾ 115, p⬍0.0001) and IL-4 (111 ⫾ 30 vs. 48 ⫾ 26 and 23 ⫾ 18, p⬍0.0001) versus untreated recipients. Only rapamycin significantly inhibited in vitro hSMC proliferation versus untreated cells (p⬍0.02). Conclusions: R348 effectively attenuates OAD by possessing immune cell specificity while preserving the respiratory epithelium in contrast to rapamycin which exerts its effects on multiple cell types. 331 Oral Administration of Nitrite or Nitrate, Abundant in the Mediterranean Diet, Attenuates Cardiac Allograft Rejection in Rats J. Zhan1,2, A. Nakao1, R. Sugimoto1, Y. Wang1, Z. Wang1, K.R. McCurry1 1Heart, Lung and Esophageal Surgery Institute, University of Pittsburgh, Pittsburgh, PA; 2Tianjin Children’s Hospital, Tianjin, China Purpose: Nitrite is a potent vasodilator and signaling molecule with cytoprotective properties. Bioconversion of dietary nitrate to nitrite by commensal oral flora has been suggested to underlie the cardiovascular protective effects of the Mediterranean diet. We hypothesized that dietary nitrite or nitrate could protect cardiac allografts. Methods and Materials: Fully allogeneic heterotopic heart transplantation (HHT) was performed in LEW to BN combination under tacrolimus (0.5 mg/kg, d0-6). Recipients were given either regular or low nitrogen oxides (NOx) chow combined with regular water or water containing nitrite or nitrate for 100 days continuously beginning 7 days before HHTx. Graft rejection was diagnosed by absence of a palpable heartbeat. Results: Supplementation of drinking water with nitrite (50 mg/L) was remarkably effective in prolonging heart graft survival and was associated with abrogation of mRNA elevation for IFN␥ in the spleen and TNF␣ in the grafts seen in untreated animals. In contrast, consumption of a low NOx diet resulted in significantly earlier rejection of allografts. Addition of nitrite water reversed the early rejection seen in low NOx diet group, suggesting a key role of nitrite in graft protection. Nitrate prolonged allograft survival in a similar manner as nitrite at low dose (200 mg/L), while high dose nitrate (1g/L) had minimal effects. Measurement of serum nitrite/nitrate
Abstracts
Results: Histopathological analysis of renal tissues demonstrated frequent arterial hyalinosis, glomerusclerosis, almost complete glomerular collapse and prominence of visceral epithelial cells that contain blebs and vacuoles. We also observed tubular changes pertaining to renal toxicity such as tubular atrophy/vacuolization, epithelial blebs, and hyalinization. Additionally, increase in the number of intratubular proteinaceous casts, proteinaceous material swollen proximal tubular epithelial cells with increased intracytoplasmic protein droplets were seen in Sirolimus treated recipients. Intrarenal mRNA expression (Mean ⫾ SEM, expressed as relative fold expression) of CTGF (125 ⫾ 18), renin (175 ⫾ 45), p22phox (400 ⫾ 216) and TGF- (85 ⫾ 12) was observed in SRL treated rat cardiac transplant recipients. Conclusions: These studies provide evidence that even a short-term course of Sirolimus exposure leads to arterial and glomerular hyalinosis protein droplets indicative of proteinuria. These changes occur in concert with activation of the RAS, oxidative stress and tissue repair pathways. 329 Simvastatin Treatment Attenuates Obliterative Airway Disease Development in Rat Tracheal Allograft Model J.O. Ropponen1,2, J.M. Tikkanen1, M. Hollmen1, R. Krebs1, M.A. Keranen1, K.B. Lemstrom1,2 1University of Helsinki, Helsinki, Finland; 2University of Helsinki, Helsinki, Finland Purpose: Retrospective analyses have implied that statin therapy has a role in the prevention of bronchiolitis obliterans syndrome (BOS). We investigated the role of simvastatin in the development of obliterative airway disease (OAD) using a rat model of OAD. Methods and Materials: In fully-mismatched non-immunosuppressed rat tracheal allografts, we used simvastatin at different doses (0.1 – 20 mg/kg orally, daily) to assess its effect on OAD development. The effects of simvastatin treatment on alloimmune activation were measured using immunohistochemistry and PCR. We also administered L-NAME to inhibit the nitric oxide (NO) pathway. Rats were harvested 3, 10 or 30 days after transplantation. Results: In tracheal allografts epithelial injury was seen 3 days after transplantation. At day 10, regeneration of the epithelium was noticed in simvastatin therapy groups but not in control group. There was no difference in OAD development between the groups at this timepoint. At day 10 simvastatin also reduced inflammation in the allografts by suppressing neutrophil accumulation. At 30 days, simvastatin therapy reduced OAD development (p⬍ 0.05) in all other doses except the lowest dose (0.1 mg/kg). L-NAME-mediated NO synthase inhibition negated partly but not totally the beneficial effects of simvastatin at 10 day and 30 day time-points. Conclusions: Simvastatin treatment effectively inhibits OAD development. According to our data, the beneficial effect of simvastatin seems to be partly NO-dependent. Together with the clinical data available, these results suggest a possible role for statins in the prevention of BOS. We are working on further evaluation of the mechanism of simvastatin beneficial effects. 330 Effects of the JAK3-Inhibitor R348 on Different Cell Types Involved in Obliterative Airway Disease J.B. Velotta1, T. Deuse1, C. Peter2, E. Masuda3, V. Taylor3, G. Park3, D. Carroll3, R.C. Robbins1, S. Schrepfer1 2Rigel Inc, South San Francisco, CA Purpose: Janus-kinase-(JAK)3 is crucial for signal transduction in immune cells.
The Journal of Heart and Lung Transplantation February 2009
Methods and Materials: Trachea from BN-donors were transplanted heterotopically into the greater omentum of Lew-rats. Recipients were treated with either R348 (40 mg/kg/d), rapamycin (3mg/kg/d), or left untreated for 28 days. Trachea specimens were evaluated histologically and tracheal epithelial mucin gene 4 (Muc4) mRNA levels were determined between all groups using quantitative real time RT-PCR. Prevention of cellular rejection was investigated between all groups using enzymeELISPOT assays.To assess the role of R348 on smooth muscle cells, platelet derived growth factor (PDGF)stimulated human smooth muscle cells (hSMCs) were incubated with each respective drug (100nM) and a MTT proliferation assay was performed. Results: R348 40 mg/kg/d treated recipients and syngeneic recipients contained moderate and outstanding levels of PAS-positive goblet cells, respectively. Both rapamycin 3 mg/kg/d and untreated recipients expressed no PAS-positive goblet cells. These histological results were confirmed by qRT-PCR that showed R348 40 mg/kg/d treated recipients had 176-fold greater Muc4 RNA levels compared to untreated recipients (1.04 ⫾ 0.9 vs. 0.0059 ⫾ 0.003, p⬍0.02) and also significantly expressed 16-fold greater Muc4 RNA levels versus rapamycin 3 mg/kg/d treated recipients (1.04 ⫾ 0.9 vs. 0.06 ⫾ 0.1, p⬍0.02). R348 40 mg/kg/d treated recipients expressed similar Muc4 RNA levels compared to syngeneic recipients. Both, R348 and rapamycin, significantly reduced the Th1 (IFN- ␥) and Th2 (IL-4) immune response as evidenced by decreased spot frequencies of IFN-␥ (1064 ⫾ 117 vs. 366 ⫾ 50 and 194 ⫾ 115, p⬍0.0001) and IL-4 (111 ⫾ 30 vs. 48 ⫾ 26 and 23 ⫾ 18, p⬍0.0001) versus untreated recipients. Only rapamycin significantly inhibited in vitro hSMC proliferation versus untreated cells (p⬍0.02). Conclusions: R348 effectively attenuates OAD by possessing immune cell specificity while preserving the respiratory epithelium in contrast to rapamycin which exerts its effects on multiple cell types. 331 Oral Administration of Nitrite or Nitrate, Abundant in the Mediterranean Diet, Attenuates Cardiac Allograft Rejection in Rats J. Zhan1,2, A. Nakao1, R. Sugimoto1, Y. Wang1, Z. Wang1, K.R. McCurry1 1Heart, Lung and Esophageal Surgery Institute, University of Pittsburgh, Pittsburgh, PA; 2Tianjin Children’s Hospital, Tianjin, China Purpose: Nitrite is a potent vasodilator and signaling molecule with cytoprotective properties. Bioconversion of dietary nitrate to nitrite by commensal oral flora has been suggested to underlie the cardiovascular protective effects of the Mediterranean diet. We hypothesized that dietary nitrite or nitrate could protect cardiac allografts. Methods and Materials: Fully allogeneic heterotopic heart transplantation (HHT) was performed in LEW to BN combination under tacrolimus (0.5 mg/kg, d0-6). Recipients were given either regular or low nitrogen oxides (NOx) chow combined with regular water or water containing nitrite or nitrate for 100 days continuously beginning 7 days before HHTx. Graft rejection was diagnosed by absence of a palpable heartbeat. Results: Supplementation of drinking water with nitrite (50 mg/L) was remarkably effective in prolonging heart graft survival and was associated with abrogation of mRNA elevation for IFN␥ in the spleen and TNF␣ in the grafts seen in untreated animals. In contrast, consumption of a low NOx diet resulted in significantly earlier rejection of allografts. Addition of nitrite water reversed the early rejection seen in low NOx diet group, suggesting a key role of nitrite in graft protection. Nitrate prolonged allograft survival in a similar manner as nitrite at low dose (200 mg/L), while high dose nitrate (1g/L) had minimal effects. Measurement of serum nitrite/nitrate