Rho-Associated Kinase Inhibitor Reduces Tumor Recurrence After Liver Transplantation in a Rat Hepatoma Model

Tumor recurrence after liver transplantation still remains a significant problem in patients with hepatocellular carcinoma. The small GTPase Rho/Rho-associated kinase (ROCK) pathway is involved in the motility and invasiveness of cancer cells. We investigated whether tacrolimus activated the Rho/ROCK signal pathway to promote the invasiveness of rat hepatocellular carcinoma cells. We also investigated whether the ROCK inhibitor Y-27632 suppressed tumor recurrence after experimental liver transplantation in a rat hepatocellular carcinoma model. Orthotopic liver transplantation was performed in hepatocellular carcinoma cell line McA-RH7777-bearing rats. Tacrolimus was administered to liver transplant rats and these rats were divided into two groups: the Y-27632-treated (10 mg/kg, for 28 days) group and the Y-27632-untreated group. Tacrolimus enhanced the cancer cell migration and stimulated phosphorylation of the myosin light chain (MLC), a downstream effector of Rho/ROCK signaling. Y-27632 suppressed the cancer cell migration and tacrolimus-induced MLC phosphorylation. Suppression of tumor recurrence after liver transplantation and significant prolongation of survival were observed in the Y-27632-treated rats in comparison with theY-27632-untreated rats. Tacrolimus stimulates the Rho/ROCK signal pathway to enhance the invasiveness of hepatocellular carcinoma, and the ROCK inhibitor Y-27632 can be used as a new antimetastatic agent for the prevention of tumor recurrence after liver transplantation.     

Reduction of hepatic ischemia/reperfusion-induced injury by a specific ROCK/Rho kinase inhibitor Y-27632

BACKGROUND: The low-molecular-weight GTPase Rho is known to act as a molecular switch by activating several downstream effectors, one of which is Rho-associated coiled-coil forming protein kinase (ROCK). ROCK/Rho kinase mediates cytoskeleton-dependent cell functions, such as actomyosin-based smooth muscle contraction and integrin-mediated cell adhesion. A specific inhibitor of ROCK/Rho kinase, Y-27632, was recently developed. The present study examined whether Y-27632 could provide a beneficial effect on hepatic ischemia/reperfusion (I/R)-induced injury through the attenuation of microcirculatory disturbance. MATERIALS AND METHODS: In male Sprague-Dawley rats, normothermic partial ischemia was induced by clamping the hepatic pedicle to the left and median lobes for 90 min, followed by 2 h of reperfusion. In the treatment group, Y-27632 was intravenously administered prior to ischemic insult. Hepatic microcirculation was investigated by using intravital fluorescence microscopy. Liver enzyme release and histological changes of the liver tissue were also evaluated. RESULTS: Y-27632 significantly improved sinusoidal perfusion and reduced the number of leukocytes sticking in hepatic sinusoids and adhering in postsinusoidal venules. The postischemic narrowing of both sinusoids and postsinusoidal venules was also markedly suppressed. Consequently, liver enzyme release was reduced and postischemic histological damage was suppressed. CONCLUSIONS: A specific ROCK/Rho kinase inhibitor, Y-27632, was useful to alleviate hepatic I/R-induced injury through ameliorating postischemic microcirculation. The administration of Y-27632 may be a novel strategy for conquering hepatic I/R-induced injury.     

Screening RhoA/ROCK inhibitors for the ability to prevent chronic rejection of mouse cardiac allografts

BackgroundChronic rejection of transplanted organs is a major obstacle in organ transplantation. The main symptoms of chronic rejection are vessel occlusion and tissue fibrosis. Macrophages play a crucial role in chronic rejection. We showed previously that RhoA deletion or RhoA/Rock inhibition using Y27632 inhibitor reorganizes macrophage actin cytoskeleton, prevents macrophage movement to the cardiac allografts, and abrogates chronic rejection in rodent models. Although besides Y27632 there are other RhoA/ROCK inhibitors available commercially, their efficacy in inhibition of chronic rejection remains unknown.MethodsWe screened four RhoA/Rock inhibitors for their ability to inhibit chronic rejection of BALB/c [H-2d] mouse cardiac allografts heterotopically transplanted into C57BL/6 [H-2b] recipients. We also tested the effect of inhibitors on macrophages in vitro.ResultsWe found that out of four tested compounds, the Fasudil and Azaindole, inhibited vessel occlusion, tissue fibrosis, decreased M2 macrophage infiltration and abrogated chronic rejection of mouse cardiac allografts. The remaining inhibitors, SAR-407899 and SLX-2119, decreased only tissue fibrosis, and were ineffective or only slightly effective in inhibiting vessel occlusion. We also found that Azaindole and Fasudil affected actin cytoskeleton and protein expression in mouse peritoneal macrophagesConclusionResults of these studies might help in development of anti-chronic rejection therapy for clinical use.     

Y-27632 prevents tubulointerstitial fibrosis in mouse kidneys with unilateral ureteral obstruction

BACKGROUND: The small GTPase Rho is involved in cell-to-substratum adhesion and cell contraction. These actions of Rho mediated by downstream Rho effectors such as Rho-associated coiled-coil forming protein kinase (ROCK) may be partly responsible for the progression of renal interstitial fibrosis. METHODS: The anti-fibrosis effects of Y-27632, a specific ROCK inhibitor, were studied both in vivo (unilateral ureteral obstruction; UUO) and in vitro. To investigate the therapeutic efficacy of Y-27632 in UUO kidneys, smooth muscle alpha actin (SMalphaA) expression, macrophage infiltration and fibrosis in the obstructed kidneys were studied. SMalphaA, transforming growth factor beta (TGF-beta), alpha1 (I) collagen, osteopontin, macrophage chemoattractant peptide-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1) gene expression were examined by Northern blotting. To elucidate the mechanism linking the Rho-ROCK pathway with renal fibrosis, the effects of Y-27632 on in vitro cell proliferation and cell migration were studied. RESULTS: In vivo analysis showed that Y-27632 suppressed SMalphaA expression, macrophage infiltration and interstitial fibrosis, and that Y-27632 suppressed SMalphaA, TGF-beta and alpha1 (I) collagen mRNA expression. In vitro analysis showed that Y-27632 did not suppress proliferation of renal fibroblasts but suppressed migration of macrophages. CONCLUSIONS: The Rho-ROCK system may play an important role in the development of tissue fibrosis, and the Rho-ROCK signaling pathway may be a new therapeutic target for preventing interstitial fibrosis in progressive renal disease.     

ROCK inhibitor Y-27632 prevents primary graft non-function caused by warm ischemia/reperfusion in rat liver transplantation

Hepatic stellate cells (HSCs) can easily be activated by ischemia/reperfusion, and this activation results in hepatic microcirculatory disturbance by cell contraction. ROCK is one of the key regulators of the motility of HSCs, and Y-27632 suppresses the activation of HSCs. We examined whether Y-27632 treatment prevents primary graft non-function caused by 45-min warm ischemia in orthotopic liver transplantation (OLT). Donor and recipient rats were administered Y-27632 (3-30 mg/kg). Y-27632 treatment at 30 mg/kg in both donor and recipient prevented congestion of the grafted livers, as demonstrated by analysis of hemoglobin (Hb) content in the grafted livers, using in-vivo near-infrared spectroscopy. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and hyaluronic acid at 4 h after OLT in the 30-mg/kg Y-27632-treated group were significantly lower than those in the control group. Specimens from the untreated control recipients showed sinusoidal congestion and massive fresh hepatocyte necrosis, whereas specimens from the Y-27632-treated recipients demonstrated minimal histological changes. Moreover, Y-27632 pre-treatment dramatically improved the survival of recipients. These results suggest that Y-27632 would be clinically useful for preventing liver failure associated with ischemia/reperfusion in liver transplantation.     

Increased expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in rat cardiac allografts

This study was designed to investigate the role of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) during chronic cardiac allograft rejection. Wistar rats were used as donors, and SD rats as recipients heterotopic cardiac transplants. Recipients pretreated with inoculation of donor splenocytes (SPC) followed by cyclophosphamide (CP) were divided into 4 groups: (A) untreated group (n = 18) without immunosuppression; (B) SPC plus CP-treated group (n = 18) that were euthanized at 15-120 days posttransplantation; (C) CsA-treated group (n = 18) euthanized at 2-3 months posttransplantation; and (D) tolerance group (n = 18) treated with SPC plus CP and monitored for at least 1 year posttransplantation. Cardiac allografts were harvested at various times for immunohistochemical studies performed to evaluate the expression of ICAM-1 and VCAM-1. Pretreatment of animals with SPC and CP induced long-term cardiac allograft survival. Immunohistochemical staining demonstrated a low level of ICAM-1 and VCAM-1 expression in cardiac allograft muscle and coronary arteries among Groups B and D. In contrast, the expressions of ICAM-1 and VCAM-1 in cardiac allografts of Groups A and C were significantly higher than those in Groups B and D. Our results suggested that the expression of ICAM-1 and VCAM-1 plays an important role during the development of chronic cardiac allograft rejection.     

Antiproliferative effects of NKH477, a forskolin derivative, on cytokine profile in rat lung allografts.

OBJECTIVE: NKH477 was recently identified as a water-soluble forskolin derivative and was reported to prolong survival of murine cardiac allografts. However, the mechanism of the efficacy is not clear in vivo. The aim of this study was to investigate the immunosuppressive effects of NKH477 on acute lung allograft rejection in the rat model and its mechanism of action in vivo. METHODS: Left lungs were transplanted orthotopically from Brown-Norway donors to Lewis recipients. Recipient rats were untreated or treated daily with different doses of NKH477. Grafts were excised on Day 3 or Day 5 to determine histopathological rejection and expressions of interleukin (IL)-2, IL-4, IL-10, and interferon (IFN)-gamma by enzyme-linked immunosorbent assay. The cytokine expression at Day 3 or Day 5 was also evaluated in recipient spleens by immunohistochemistry. Furthermore, mesenteric lymph node cells from recipients at Day 5 were cultured alone or stimulated with donor antigens for 72 hours to determine cell proliferation by means of thymidine incorporation. RESULTS: NKH477 significantly extended allograft survival time in a dose-dependent manner and reduced histopathological rejection. Treatment with NKH477 inhibited IFN-gamma and IL-10 expression, whereas expression of these cytokines were markedly upregulated in the untreated allografts. Expression of IL-2 and IL-10 also increased in the spleen of untreated allorecipients. NKH477 suppressed expression of both cytokines in the spleen. In addition, lymphocyte proliferation was inhibited in NKH477-treated recipients as compared with untreated recipients. CONCLUSION: These results suggest that NKH477 exerts an antiproliferative effect on lymphocytes in vivo with an altered cytokine profile in rat recipients of lung allografts.     

Distinct requirements for host CD80/CD86 costimulatory molecules in cardiac versus islet rejection

The role of B7 family members CD80 and CD86 in providing costimulatory signals to T cells is well established. Interestingly, previous studies show that host CD80/CD86 expression is required for cardiac allograft rejection. However, the role for host costimulation by CD80/CD86 molecules for the rejection of neovascularized islet allografts and xenografts is unknown. The purpose of this study was to determine whether islet allografts and/or rat islet xenografts required host CD80/CD86 molecules for acute rejection. Streptozotocin-induced diabetic C57Bl/6 (B6, H-2(b)) or B6 CD80/CD86 double-deficient mice were grafted with allogeneic BALB/c (H-2(d)) islet allografts or with WF (RT1(u)) islet xenografts. Nondiabetic B6 mice were grafted with BALB/c heterotopic cardiac allografts. Consistent with previous reports, BALB/c islet allografts were acutely rejected in wild-type B6 mice could survive long-term (>100 days) in B6 CD80/CD86-deficient animals. In stark contrast, both islet allografts and WF rat islet xenografts demonstrated acute rejection in both control B6 and in B6 CD80/CD86 deficient hosts. In conclusion, varied studies imply that the inherent pathways for rejecting primarily vascularized versus cellular allografts or xenografts may be distinct. The present study illustrates this concept by showing a marked difference in the role of host-derived CD80/CD86 costimulatory molecules for cardiac allograft versus islet allograft/xenograft rejection in vivo. Although such costimulation is rate limiting for cardiac allograft rejection, these same molecules are not necessary for acute rejection of either islet allografts or xenografts.     

Organ-specific differences in the function of MCP-1 and CXCR3 during cardiac and skin allograft rejection

BACKGROUND: Chemokines are well-established to function in the recruitment of leukocytes into allografts in the course of rejection. Moreover, some studies have indicated that there are organ-specific differences in chemokine function, but the mechanism accounting for this difference is not known. METHODS: Fully major histocompatibility complex-mismatched vascularized cardiac transplants or skin transplants were performed using BALB/c (H-2d), C57BL/6 (H-2b), MCP-1-/- (H-2b) and CXCR3-/- (H-2b) mice as donors or recipients. Also, skin grafts (H-2b) were placed onto SCID mice (H-2d) that received BALB/c splenocytes (H-2d) by adoptive transfer either at the time of transplantation, or after a period of 28 days. RESULTS: Cardiac allografts in MCP-1-/- recipients survived significantly longer (P<0.0005) than wild-type (WT) controls. However, there was no prolongation of survival when MCP-1-/- grafts were used a donors in WT mice. In contrast, the absence of donor but not recipient MCP-1 prolonged skin allograft survival. WT donor cardiac grafts in CXCR3-/- recipients had a modest prolongation of survival (P<0.0005), whereas CXCR3-/- donor cardiac grafts in WT recipients were rejected similar to controls. Also, while recipient CXCR3 had no effect on the rejection of skin, CXCR3-/- donor skin grafts survived significantly longer than WT controls. This survival advantage was lost when vascularized CXCR3-/- skin grafts were used as donors in the SCID model of rejection. CONCLUSION: Recipient derived MCP-1 and CXCR3 are functional in the rejection of vascularized, but not nonvascularized, allografts. In contrast, donor-derived MCP-1 and CXCR3 are functional in nonvascularized, but not vascularized grafts.     

Donor and recipient contributions of ICAM-1 and P-selectin in parenchymal rejection and graft arteriosclerosis: insights from double knockout mice.

BACKGROUND: Mice with target gene deletions were used in an immunosuppressed, heterotopic mouse cardiac transplant model to investigate the effects of simultaneous deficiencies of ICAM-1 and P-selectin on late cardiac rejection. METHODS: To determine the contribution of donor sources of ICAM-1 and P-selectin, ICAM-1/P-selectin gene deficient (I/P -/-) (n = 7) or wild type (n = 6) donor hearts were placed into CBA recipients. To study recipient sources of ICAM-1 and P-selectin, wild type donor hearts were placed into I/P -/- (n = 7) or wild type (n = 13) recipients. Recipients received a 30-day course of anti-CD4/8 mAb. RESULTS: I/P -/- donor allografts had prolonged survival (52-57 days) compared with wild type allografts (49-51 days). I/P -/- donor allografts underwent parenchymal rejection with mononuclear cell infiltration and developed alpha-smooth muscle actin positive vascular thickening (30 +/- 7% luminal occlusion, n = 78 vessels). Wild type allografts had parenchymal rejection with vascular medial necrosis and an absence of arteriosclerotic thickening (10 +/- 8%, n = 75, p = 0.008). Using the reverse combination, allografts from I/P -/- or wild type recipients had similar graft survival (50-57 days), comparable but variable degrees of parenchymal rejection, and comparable vascular occlusion (22 +/- 15% vs 28 +/- 19%, p = 0.442). CONCLUSION: We have shown that donor and recipient sources of ICAM-1 and P-selectin may have independent roles in leukocyte trafficking to the graft. Simultaneous interruption of donor ICAM-1 and P-selectin delays onset of parenchymal rejection. However, donor I/P deficiency permits arteriosclerotic development, perhaps by attenuating the alloimmune injury. In contrast, recipient deficiency alone does not altergraft outcomes suggesting that the donor is the critical site of ICAM-1 and P-selectin.     

Blocking of mononuclear cell accumulation, cytokine production, and endothelial activation within rat cardiac allografts by CD4 monoclonal antibody therapy.

CD4 monoclonal antibody therapy prolongs allograft survival in a variety of experimental models and is currently undergoing clinical trials, though surprisingly little is known about the effects of CD4 mAb therapy on intragraft effector mechanisms that mediate rejection. We previously reported the significantly improved survival of (LEWxBN)F1 cardiac allografts in LEW rats treated for 10 days with the new CD4 mAb, BWH-4, at a dose of 700 micrograms/day, i.v., starting at the time of engraftment. Thus, CD4-treated rats showed prolongation of allograft survival to a median of 37 days (range 22 to greater than 100 days) post-Tx, compared with rejection at 7 days in untreated controls. We now report the results of detailed immunohistologic studies of allografts collected from these rats. Comparison of acutely rejecting allografts in untreated rats with well-functioning allografts collected at day 7 post-Tx from CD4-treated rats showed that CD4 mAb: (1) significantly reduced mononuclear cell infiltration, interstitial edema, hemorrhage formation and vascular and extravascular thrombosis; (2) inhibited mononuclear cell induction of receptors for IL-2 and transferrin, and upregulation of class II antigens and ICAM-1 on leukocytes and endothelial cells; (3) suppressed intragraft mononuclear cell and/or endothelial production of the cytokines IL-1, IL-2, IL-6, IFN-gamma, and TNF; and (4) blocked upregulation of endothelial tissue factor and downregulation of thrombomodulin, and consequently inhibited fibrin deposition. Studies of allografts from CD4-treated rats collected at day 30 post-Tx, prior to clinical rejection, showed a resurgence of CD4+ cells within allografts and a dense cellular immune response. We conclude that short-term CD4 mAb therapy has potent and extensive inhibitory effects on cytokine-related mononuclear cell and endothelial activation in vivo, blocking multiple afferent and efferent steps of the alloresponse.     

Acute rejection and cardiac graft vasculopathy in the absence of donor-derived ICAM-1 or P-selectin.

BACKGROUND: ICAM-1 and P-selectin are molecules that facilitate adhesion of circulating leukocytes to vessel walls. We have investigated the role of donor-derived ICAM-1 and P-selectin in acute and chronic cardiac allograft rejection. METHODS: C57BL/6J (H-2(b)) mice were used as donors for heterotopic heart transplantation into CBA/Ca (H-2(k)) recipients. The donors were wild-type or homozygous for gene mutations of ICAM-1 or P-selectin. We measured acute rejection in non-immunosuppressed recipients by daily palpation and sacrificed mice at Days 2, 4, and 6 for immunohistochemical analysis. For chronic rejection, recipients received monoclonal antibody against CD4+ T cells. We removed hearts at Days 60 to 62 for histologic assessment of vasculopathy using quantitative morphometry to measure intimal thickening. RESULTS: Time (days) to rejection was 7.1 +/- 0.57 for wild-type (n = 10), 7.0 +/- 0.71 for ICAM-1 -/- (not significantly different, n = 7) and 6.1 +/- 0.33 (p = 0.001) for P-selectin -/- donors. ICAM-1 deficiency was associated with delayed infiltrate at Day 4 compared with wild-type. In the model of chronic rejection, elastin-positive vessels showed a mean occlusion of 34% +/- 3% in transplanted wild-type hearts; vessels were divided into those showing 0% to 20%, 20% to 50%, and 50% to 100% occlusion. We observed no difference in the number of affected vessels or the amount of vascular thickening in donors lacking ICAM-1 or P-selectin compared with wild-type controls. CONCLUSIONS: The absence of ICAM-1 or P-selectin in donor tissues neither lengthens the time of allograft survival nor inhibits the vascular lesions associated with chronic rejection. Indeed, the absence of P-selectin may exacerbate alloimmune injury.     

Stimulation of Ectopic Bone Formation in Response to BMP-2 by Rho Kinase Inhibitor: A Pilot Study

The small GTPase Rho and Rho-associated protein kinase (Rho kinase, ROCK) signal participates in a variety of biological functions including vascular contraction, tumor invasion, and penile erection. Evidence also suggests Rho-ROCK is involved in signaling for mesenchymal cellular differentiation. However, whether it is involved in osteoblastic differentiation is unknown. We therefore asked whether Rho-ROCK signaling participates in recombinant human bone morphogenetic protein (rhBMP-2)-induced osteogenesis both in vitro and in vivo. Continuous delivery of a specific ROCK inhibitor (Y-27632) enhanced ectopic bone formation induced by rhBMP-2 impregnated into an atelocollagen carrier in mice without affecting systemic bone metabolism. Treatment with Y-27632 also enhanced the osteoblastic differentiation of cultured murine neonatal calvarial cells. These effects were associated with increased expression of BMP-4 gene. Expression of a dominant negative mutant of ROCK in ST2 cells promoted osteoblastic differentiation, while a constitutively active mutant of ROCK attenuated osteoblastic differentiation and the ROCK inhibitor reversed this phenotype. Thus, ROCK inhibits osteogenesis, and a ROCK inhibitor in combination with the local delivery of rhBMP/collagen composite may be clinically applicable for stimulating bone formation.     

Detection of an up-regulation of a group of chemokine genes in murine cardiac allograft in the absence of interferon-gamma by means of DNA microarray

BACKGOUND: Interferon (IFN)-gamma and the IFN-gamma-dependent pathway are prominent in vascularized allograft during acute rejection. However, IFN-gamma deficient (IFN-gamma-/-) mice can rapidly reject cardiac allografts. To bring the alternative pathway during allograft rejection into more precise focus, we investigated the gene expression profile in murine cardiac allografts in IFN-gamma-/- mice by means of DNA microarray. MATERIAL AND METHOD: We screened for gene expression changes in murine cardiac allografts of BALB/c H-2d into both wild-type C57BL/6 H-2b (n=3) and IFN-gamma-/- C57BL/6 H-2b(IFN-gamma-/-, n=4) using Affymetrix oligonucleotide arrays to monitor more than 11,000 genes and expressed sequence tag (ESTs). The heart was heterotopically transplanted. Transplanted hearts were harvested on day 5. As a control, isografts (C57BL/6 to C57BL/6) were also harvested on day 5. RESULTS: On day 5, 64 of the 84 genes induced in the allografts in wild-type mice were not up-regulated in IFN-gamma-/- mice. We identified a group of 54 genes that were up-regulated in allografts in IFN-gamma-/- mice. Several chemokine genes, including monocyte chemoattractant protein=1 and macrophage inflammatory protein, were induced in the allografts in both wild-type and IFN-gamma-/- mice. Interestingly, a group of genes, including C10-like chemokine and platelet factor 4, were specifically induced in the IFN-gamma-/- mice. CONCLUSION: DNA microarray analysis reveals a unique pattern of mRNA expression in allografts in IFN-gamma-/- mice as well as a group of genes induced in cardiac allografts in both wild-type and IFN-gamma-/- mice, including monocyte chemoattractant protein-1 and monocyte chemoattractant protein-1.     

alpha4 integrin in islet allograft rejection.

BACKGROUND: Adhesion molecules are involved in multiple steps of the continuum of allograft rejection. We studied the effects of blockade of the interactions between alpha4 integrin and its ligands, vascular cell adhesion molecule-1 (VCAM-1) and fibronectin, on allograft survival. METHODS: Streptozotocin-induced diabetic CBA (H-2k) mice received islet transplants from BALB/c (H-2d) donors. Recipient mice were treated with antibodies against alpha4 integrin (PS/2), VCAM-1 (MK 2.7), and a peptide corresponding to the binding site of alpha4 integrin on fibronectin (connecting segment 1 peptide, CS1-peptide). Graft function was measured by daily tail vein blood glucose levels, with rejection defined as the return of hyperglycemia. Graft-bearing kidneys were removed for immunohistochemical analysis. RESULTS: Treatment with anti-alpha4 integrin antibody, anti-VCAM-1 antibody, or with CS1-peptide led to long-term survival of islet allografts. Recipients with long-surviving islet grafts did not show tolerance, in that they rejected a second donor-type islet allograft. Although both anti-alpha4 integrin antibody and CS1-peptide completely abolished cellular infiltration of the islet graft 7 days after transplantation, anti-VCAM-1-treated recipients showed a dense peri-islet infiltrate of activated, alpha4 integrin+, cytotoxic T cells. CONCLUSIONS: These data show that alpha4 integrin is critically important to allograft rejection. Anti-VCAM-1 antibody appears to prevent rejection without qualitatively affecting either T cell activation or migration to the graft. Conversely, anti-alpha4 integrin antibody and CS1-peptide may prevent islet allograft rejection by altering either T cell activation or lymphocyte trafficking. Blocking interactions between alpha4 integrin and its ligands may provide novel forms of immunosuppression.     

A comparison of gene expression in murine cardiac allografts and isografts by means DNA microarray analysis

BACKGROUND: Acute rejection of allografts remains a significant problem in clinical transplantation, and the fundamental mechanism underlying this rejection are as yet only poorly elucidated. Recently, DNA microarrays have come into use for the study of gene expression profiles, and we have taken advantage of this new technology to investigate acute rejection. We compared mRNA profiles in murine cardiac allografts with isografts using DNA microarrays with probe sets corresponding to more than 11,000 mice genes. METHODS: We screened for gene expression changes in murine cardiac allografts between fully incompatible mice strains (BALB/c H2d to C3H/He H2k) using a DNA microarray. The heart was heterotopically transplanted. Allografts (BALB/c to C3H/He) were removed on days 1, 3, and 5. As a control, isografts (C3H/He to C3H/He) harvested on days 1, 3, and 5 and native hearts of both strain mice (C3H/He and BALB/c) were obtained. RESULTS: On day 5, interferon-gamma (IFN-gamma) and many IFN-gamma-inducible genes were profoundly induced in the allograft relative to isograft. Monokine induced by IFN-gamma was most profoundly induced followed by inducibly expressed GTPase and Lmp-2. IFN-gamma was also profoundly induced. The induction was detectable from day 3. In contrast, genes regulated by other cytokines exhibited only modest changes. CONCLUSION: IFN-gamma-inducible genes are specifically up-regulated in murine cardiac allografts, suggesting that signaling mediated by IFN-gamma may play an important role in the late phase of acute rejection in vivo.     

The contribution of adhesion molecule expression in donor kidney biopsies to early allograft dysfunction.

BACKGROUND: Renal allograft rejection is associated with the expression of adhesion molecules on vascular endothelial and tubular epithelial cells. METHODS: To assess whether the number of cell adhesion molecules expressed in donor kidneys can predict early rejection or delayed graft function, kidney biopsies from 20 living and 53 cadaveric kidney donors were obtained before engraftment into the recipients and the expression of the cell adhesion molecules intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and endothelial leukocyte adhesion molecule (E-selectin) were determined by immunohistochemistry. RESULTS: All biopsies from living donors showed significantly lower expression of ICAM-1 and VCAM-1 compared to biopsies from cadaveric donors. There was no difference in the expression of adhesion molecules on tubular cells between transplants with primary function compared to allografts with early rejection in living donated kidneys (ICAM-1: 2+/-8 vs. 3+/-8%; VCAM-1: 9+/-7 vs. 1+/-1%), as well as in cadaveric kidneys (ICAM-1: 38+/-29 vs. 39+/-38%; VCAM-1: 55+/-27 vs. 48+/-29%). The expression of ICAM-1 molecules on tubular cells was determined to be a predictor for the occurrence of delayed graft function in cadaveric kidneys (ICAM-1: 65+/-24* vs. 38+/-29% delayed graft versus primary graft function). No delayed graft function occurred in recipients of living donated kidneys. CONCLUSIONS: These data suggest that adhesion molecule expression in donor biopsies is not a predictor for early allograft rejection, but can be used as a marker for the development of postischemic acute renal allograft failure.     

microRNA-93靶向调控Rho相关激酶2/LIMK/Cofilin通路影响骨癌痛小鼠痛行为

目的 观察microRNA-93(miR-93)及Rho相关激酶(rho associated coiled-coil forming protein kinase,ROCK)通路对小鼠骨癌痛(bone cancer pain,BCP)行为的影响并探讨其潜在机制.方法 将C57BL/6J小鼠采用随机数字表法分为对照组(Control组)、假手术组(Sham组)及BCP组、BCP+PBS组、BCP+Y-27632(ROCK抑制剂)组,每组13只.建模前1d及建模后3、5、7、10、14 d进行动物痛行为学评估,观察小鼠对机械和热刺激的反应.建模后第14天处死小鼠,取术侧L4～L6背根神经节(dorsal root ganglion,DRG),SYBR Green实时荧光定量PCR法观察miR-93表达变化,双荧光素酶实验检测miR-93对ROCK2基因的靶向调控.Western blot法观察ROCK2、LIMK(LIM domain kinase,LIMK)和Cofilin蛋白在DRG神经元中表达.结果 C57BU6J小鼠股骨接种NCTC2472细胞后,患侧后肢分别在第5、7天开始出现明显的触诱发痛[Sham组(6.81±1.04)g,BCP组(5.12±0.47)g]和热痛过敏[Sham组(11.75±1.46)s,BCP组(7.61±1.07)s],并呈进行性加重(P＜0.05);BCP组小鼠miR-93表达随疼痛加重呈时间依赖性降低,而ROCK2表达呈时间依赖性增加(P＜0.05),进而激活LIMK/Cofilin通路.双荧光素酶实验证实miR-93可与ROCK2 mRNA 3'-UTR直接结合,从而发挥对ROCK2转录后翻译的抑制作用(P＜0.05).鞘内注射ROCK抑制剂能有效缓解小鼠触诱发痛[BCP+PBS组(3.43±0.89)g,BCP+Y-27632组(5.74±1.02)g]和热痛过敏[BCP+PBS组(5.48±1.03)s,BCP+Y-27632组(9.82±1.24)s],同时抑制通路主要因子ROCK2、磷酸化-LIMK (phospho-LIMK,p-LIMK)、磷酸化-Cofilin (phospho-Cofilin,p-Cofilin)的表达(P＜0.05).结论 小鼠BCP疼痛过程中miR-93表达降低,进而激活ROCK2/LIMK/Cofilin通路,鞘内注射ROCK抑制剂可抑制该通路的活化,缓解小鼠BCP症状.     

Rho/Rho激酶对肝癌细胞体外侵袭转移作用的研究

目的　观察Rho/Rho激酶对恶性肿瘤细胞侵袭力的影响,探讨Rho/Rho激酶抑制剂用于肿瘤转移治疗的可能性。方法　肝癌细胞株SMMC772 1经Rho/Rho激酶抑制剂Y 2 76 32处理后,分别用黏附基质分析、重建基底膜侵袭模型以及Westernblot分析对肿瘤细胞侵袭、黏附、运动能力的影响。结果　SMMC772 1细胞有Rho蛋白表达,经Y 2 76 32处理后,细胞的侵袭能力明显下降,黏附率降低,运动能力下降。结论　Rho/Rho激酶对肝癌细胞体外侵袭转移有重要作用,Y 2 76 32可降低恶性肿瘤细胞的侵袭能力,改变侵袭相关性质     

Prevention of ischemia-reperfusion-induced hepatic microcirculatory disruption by inhibiting stellate cell contraction using rock inhibitor

BACKGROUND: We demonstrated that hepatic stellate cells (HSCs) isolated from rat livers exposed to warm ischemia are significantly contractile when compared with HSCs from intact rat livers. This suggests that ischemia-reperfusion (IR)-induced impairment of sinusoidal microcirculation results, at least in part, from contraction of HSCs. METHODS: Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) is one of the key regulators of HSCs motility. Therefore we investigated whether Y-27632, a p160ROCK-specific inhibitor, has beneficial effects on warm IR injury in an in vivo rat partial liver IR model and a rat orthotopic liver transplantation model. RESULTS: After reperfusion following 90 min of warm ischemia, livers in untreated control rats had persistent congestion and impaired mitochondrial respiration, as demonstrated by increasing deoxy-hemoglobin and reduced cytochrome oxidase contents in the hepatic tissues using in vivo near-infrared spectroscopy. Serum levels of transaminase and endothelin (ET)-1 in these rats were markedly increased 1 hr after reperfusion. In contrast, when Y-27632 (3-30 mg/kg) was administered orally, hepatic tissue contents of deoxy-hemoglobin and cytochrome oxidase rapidly normalized. In such animals, the elevation of serum transaminase levels, but not that of ET-1 levels, was significantly suppressed. This is consistent with in vitro data demonstrating that Y-27632 causes HSCs to undergo relaxation even in the presence of ET-1. Moreover, in a rat orthotopic liver transplantation model, Y-27632 pretreatment dramatically improved the survival of recipients with liver grafts subjected to 45 min of warm ischemia. CONCLUSIONS: Y-27632 attenuates IR-induced hepatic microcirculation disruption by inhibiting contraction of HSCs.