Inhibition of heat shock protein 90 attenuates post­angioplasty intimal hyperplasia.

Intimal hyperplasia (IH) is a pathologic process that leads to restenosis after treatment for peripheral arterial disease. Heat shock protein 90 (HSP90) is a molecular chaperone that regulates protein maturation. Activation of HSP90 results in increased cell migration and proliferation. 17­N­allylamino­17­demethoxygeldanamycin (17­AAG) and 17­dimethylaminoethylamino­17­demethoxygeldanamycin (17­DMAG) are low toxicity Food and Drug Association approved HSP90 inhibitors. The current study hypothesized that HSP90 inhibition was predicted to reduce vascular smooth muscle cell (VSMC) migration and proliferation. In addition, localized HSP90 inhibition may inhibit post­angioplasty IH formation. For proliferation, VSMCs were treated with serum­free media (SFM), 17­DMAG or 17­AAG. The selected proliferative agents were SFM, platelet derived growth factor (PDGF) or fibronectin. After three days, proliferation was measured. For migration, VSMCs were treated with SFM, 17­AAG or 17­DMAG with SFM, PDGF or fibronectin as chemoattractants. Balloon injury to the carotid artery was performed in rats. The groups included in the present study were the control, saline control, 17­DMAG in 20% pluronic gel delivered topically to the adventitia or intraluminal delivery of 17­DMAG. After 14 days, arteries were fixed and sectioned for morphometric analysis. Data was analyzed using ANOVA or a student's t­test. P<0.05 was considered to indicate a statistically significant difference. The results revealed that 17­AAG and 17­DMAG had no effect on cell viability. PDGF and fibronectin also increased VSMC proliferation and migration. Furthermore, both 17­AAG and 17­DMAG decreased cell migration and proliferation in all agonists. Topical adventitial treatment with 17­DMAG after balloon arterial injury reduced IH. HSP90 inhibitors suppressed VSMC proliferation and migration without affecting cell viability. Topical treatment with a HSP90 inhibitor (DMAG) decreased IH formation after arterial injury. It was concluded that 17­DMAG may be utilized as an effective therapy to prevent restenosis after revascularization.

Practicing metacognition through the use of repeated posttest predictions.

BACKGROUND: Development of metacognitive skills is one method to increase self-awareness of pharmacy students. This study sought to assess students' posttest prediction (postdiction) of performance on a series of multiple-choice examinations to determine if feedback regarding predicted and actual performance could improve personal predictive abilities over time. IMPACT: While there was a statistically significant change in the students' predictive abilities from examination one to examination three, lower scores in examination two disrupted the trend we had hoped to see. When broken down by overall course score, the highest performing students rarely overestimated their score (5-21% of the time, depending on examination), while the lowest performing students were more varied (22-56% over prediction, depending on examination). RECOMMENDATIONS: This study used a novel assessment method of postdictions without additional data points such as predictions or grade point average (GPA), which could have helped confirm the value of the method. Additionally, we realized assessing the impact of the qualitative feedback students received could elucidate why and recommend this for future studies. DISCUSSION: While students were generally poor predictors of their performance, repeated use of this skill helped them to reduce the number of over predictions made by the end of the course. This change was greatest for the lowest performing students indicating that they may receive more benefit from this exercise than higher performing students. This method of using postdictions adds to the collection of tools that can be used to measure student metacognitive skills.

Intraluminal Delivery of Simvastatin Attenuates Intimal Hyperplasia After Arterial Injury.

INTRODUCTION: Oral statins reduce intimal hyperplasia (IH) after arterial injury by only ∼25%. Alternative drug delivery systems have gained attention as carriers for hydrophobic drugs. We studied the effects of simvastatin (free vs hyaluronic acid-tagged polysialic acid-polycaprolactone micelles) on vascular smooth muscle cell (VSMC) migration, VSMC proliferation and intimal hyperplasia. We hypothesized both free and micelle containing simvastatin would inhibit VSMC chemotaxis and proliferation, and local statin treatment would be more effective than oral in reducing IH in rats following carotid balloon injury. METHODS: VSMCs pretreated with free simvastatin (20 minutes or 20 hours) or simvastatin-loaded micelles underwent chemotaxis and proliferation to platelet-derived growth factor. Next, rats that underwent balloon injury of the common carotid artery received statin therapy-intraluminal simvastatin-loaded micelles prior to injury, periadventitial pluronic gel following injury, or combinations of gel, micelle, and oral simvastatin. After 14 days, morphometric analysis determined the -intimal to medial ratio. Findings were compared to controls receiving oral simvastatin or no statin therapy. Statistical analysis was by analysis of variance for the in vitro experiments and a factorial general linear model for the in vivo experiments. RESULTS: The simvastatin-loaded micelles and free simvastatin inhibited VSMC chemotaxis (54%-60%). IH was induced in all injured vessels. Simvastatin in pluronic gel or micelles reduced IH compared to untreated controls (0.208 ± 0.04 or 0.160 ± 0.03 vs 0.350 ± 0.03, respectively); however, neither gel nor simvastatin-loaded micelles were superior to oral statins (0.261 ± 0.03). Addition of oral statins or combining both local therapies did not provide additional benefit. Micelles were the single greatest contributing factor in IH attenuation. CONCLUSIONS: Intraluminally or topically delivered statins reduced IH. The efficacy of single-dose, locally delivered statin alone may lead to novel treatments to prevent IH. The different routes of administration may allow for treatment during endovascular procedures, without the need for systemic therapy.

Fluvastatin inhibits intimal hyperplasia in wild-type but not Thbs1-null mice.

BACKGROUND: Thrombospondin-1 (TSP-1) is functionally important to intimal hyperplasia (IH) development. Statin drugs have beneficial pleiotropic effects, including reduced IH; however, the effect of statins on IH in a TSP-1-independent setting is unknown. HYPOTHESIS: Statins will be less effective in attenuating IH after vascular injury in TSP-1-null (Thbs1-/-) mice compared with wild-type (WT) mice. MATERIALS AND METHODS: Carotid artery ligation was performed on WT and Thbs1-/- mice. Each strain was divided into two groups: no statin control or standard chow containing fluvastatin (10 or 40 mg/kg/d). After 28 d, analysis included morphometric analysis and real-time quantitative reverse transcription polymerase chain reaction on the arteries and enzyme-linked immunosorbent assay on plasma (TSP-1 WT, TSP-2 WT, and Thbs1-/-). Comparisons were made by analysis of variance, with P < 0.05 considered significant. RESULTS: In no statin controls, WT mice had more IH than Thbs1-/- mice (0.46 ± 0.09 versus 0.15 ± 0.04). Fluvastatin reduced IH in the WT (0.46 ± 0.09 versus 0.23 ± 0.06), but not in Thbs1-/- groups (0.15 ± 0.04 versus 0.22 ± 0.07). No difference in IH existed between Thbs1-/- no statin controls and fluvastatin WT and Thbs1-/- groups. Statin dose did not affect IH. TSP-1 plasma levels were increased in fluvastatin WT. TSP-2 levels were decreased in fluvastatin WT and elevated in fluvastatin Thbs1-/-. Fluvastatin had no effect on tissue Thbs1 or Thbs2 gene expression. CONCLUSIONS: TSP-1 is necessary for robust IH after arterial injury. Because fluvastatin had no effect on IH in Thbs1-/-, the data suggest that the statin effect on IH may be largely TSP-1 dependent. Both statins and the presence of TSP-1 affect TSP-1 and TSP-2 plasma levels.

Green tea extract prolongs allograft survival as an adjunctive therapy along with low dose cyclosporine A.

The current immunosuppressive drugs are successful in prolonging allograft survival but fail to achieve transplantation tolerance or prevent chronic rejection. Consequently, there is ongoing research to develop novel combinatorial therapies that are more efficacious in prolonging allograft survival as well as induce tolerance toward the transplanted organ. The present study aims to study the efficacy of green tea extract (GTE) in combination with low dose cyclosporine A (CyA) in prolonging allograft survival in mice. Numerous studies have reported the anti-inflammatory and immunomodulatory properties of GTE and its various catechin components. GTE is also known to attenuate CyA induced nephrotoxicity. Therefore, we hypothesized that GTE alone or in combination with CyA will prolong graft survival. Our study demonstrates that GTE in combination with low dose CyA significantly prolongs graft survival as well as increase the production of immunosuppressive cytokine, IL-10. GTE also decreases CyA induced high TGF-beta production, which is incriminated in CyA induced nephrotoxicity. We also observed that GTE inhibits both nonspecific and antigen-specific proliferation of T cells in vitro. These results indicate the potential of GTE as an adjunctive therapy in combination with CyA to prolong allograft survival and to reduce CyA induced nephrotoxicity.

Cyclosporine inhibition of angiogenesis involves the transcription factor HESR1.

PURPOSE: Angiogenesis is critical in normal development and in tumor growth. Experimentally, cyclosporine A (CyA) inhibits angiogenesis in an in vivo mouse model and an in vitro capillary tube model. The mechanisms behind its antiangiogenic effects are not well characterized. To determine which nuclear factor, if any, may be involved in the antiangiogenic effects of CyA, we performed a microarray analysis of human aortic endothelial cells (HAEC) subjected to CyA and another calcineurin inhibitor, FK 506. METHODS: HAEC were divided into four groups: (1) HAEC incubated with CyA 2 microg/mL; (2) HAEC incubated with CyA 10 microg/mL; (3) HAEC incubated with FK 506 1 microg/mLl for 24 h; and (4) HAEC as control. We used Affymetrix GeneChip U133-A for gene expression analysis and validated our results with quantitative reverse transcription-polymerase chain reaction. RESULTS: At a 2 microg/mL dose, CyA treated HAEC revealed a 44-fold increase in the expression of hairy enhancer of split-related protein 1 (HESR1) and 1.73-fold down-regulation of transcripts encoding for the vascular endothelial growth factor (VEGF) receptor (VEGFR2). At 10 microg/mL, the expression of the HESR1 transcript was 57-fold higher than control, and VEGFR2 exhibited a 1.93-fold down-regulation. Quantitative reverse transcription-polymerase chain reaction confirmed a significant (P < 0.0001) increase in expression of HESR1 in CyA treated cells. In contrast, the expression level of HESR1 was not affected by the FK 506 treatment. CONCLUSION: CyA demonstrate antiangiogenic activities linked to an overexpression of HESR1 transcription factor, and down-regulation of VEGFR2. Thus, use of high-dose CyA may provide a novel treatment in angiogenesis dependent disease.

An interleukin-6-neutralizing antibody prevents cyclosporine-induced nephrotoxicity in mice.

INTRODUCTION: Chronic use of cyclosporine A (CyA) induces nephrotoxicity primarily due to endothelial dysfunction. In our previous studies, potential mechanisms were identified in vitro and implicated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and interleukin-6 (IL-6) as key components in causing endothelial dysfunction. In this study, we tested the hypothesis that NADPH oxidase activity and IL-6 are key components in renal damage in an in vivo model. METHODS: Male mice C57B/6 mice from Jackson Laboratory (Bar Harbor, ME) at 6-8 wks were subjected to a low-salt diet throughout the trial. After 1 week on a low-salt diet, the mice were injected daily with treatments in 50 muL vehicle composed of 75% cremaphor (Sigma, St. Louis, MO) and ethanol for 5 wks. A vehicle-alone group was also set aside. Mice were weighed and 25 mg/kg/day cyclosporine (Novartis Pharma, St. Louis, MO) was injected daily. Apocynin (Calbiochem, Gibbstown, NJ) 20 mg/kg were injected either alone or concomitantly with CyA. Another group of mice were administered IL-6 antibody (Cat no. MAB406; R&D Systems, Minneapolis, MN) at 2 mug/day along with CyA. The kidneys were removed en bloc immediately and submitted in formalin for paraffin sections. Trichrome stains were performed. Slides were blinded and 10 photographs of cortical areas per treatment group were taken, which covered an estimate of 10% surface area in random fashion. Areas of renal damage, which were determined by tubular necrosis, were identified and quantified by amount of necrosis per photograph. Each photograph was divided into 10 blocks, and the number of blocks that contained necrotic tubules per photo was recorded. RESULTS: The two control mice (low salt only) had no damage. The four vehicle mice had trace amounts of tubular necrosis. CyA treatment group demonstrated the highest amount of damage (29/70; 41%). CyA with apocynin, a specific NADPH oxidase inhibitor, was found to have 36% (22/60) damage, whereas the CyA with IL-6 antibody only was observed to have 15% (6/40) damage. Comparing imaging analysis, there was no difference between mice treated with CyA alone and with CyA with apocynin. However, the amount of damage in mice treated with CyA and IL-6 antibody was found to be significantly lower than both CyA and CyA with apocynin. CONCLUSIONS: CyA action as a calcineurin inhibitor has allowed prolongation of kidney transplants, but its chronic use has led to devastating consequences such as allograft nephropathy. Previously, we have identified potential mechanisms of CyA-induced endothelial dysfunction in vitro. The current study identifies increased IL-6 expression as a mechanism by which CyA induces renal damage and that the use of an IL-6-neutralizing antibody may be useful in reducing CyA-induced renal damage.

Ginger extract inhibits LPS induced macrophage activation and function.

BACKGROUND: Macrophages play a dual role in host defence. They act as the first line of defence by mounting an inflammatory response to antigen exposure and also act as antigen presenting cells and initiate the adaptive immune response. They are also the primary infiltrating cells at the site of inflammation. Inhibition of macrophage activation is one of the possible approaches towards modulating inflammation. Both conventional and alternative approaches are being studied in this regard. Ginger, an herbal product with broad anti inflammatory actions, is used as an alternative medicine in a number of inflammatory conditions like rheumatic disorders. In the present study we examined the effect of ginger extract on macrophage activation in the presence of LPS stimulation. METHODS: Murine peritoneal macrophages were stimulated by LPS in presence or absence of ginger extract and production of proinflammatory cytokines and chemokines were observed. We also studied the effect of ginger extract on the LPS induced expression of MHC II, B7.1, B7.2 and CD40 molecules. We also studied the antigen presenting function of ginger extract treated macrophages by primary mixed lymphocyte reaction. RESULTS: We observed that ginger extract inhibited IL-12, TNF-alpha, IL-1beta (pro inflammatory cytokines) and RANTES, MCP-1 (pro inflammatory chemokines) production in LPS stimulated macrophages. Ginger extract also down regulated the expression of B7.1, B7.2 and MHC class II molecules. In addition ginger extract negatively affected the antigen presenting function of macrophages and we observed a significant reduction in T cell proliferation in response to allostimulation, when ginger extract treated macrophages were used as APCs. A significant decrease in IFN-gamma and IL-2 production by T cells in response to allostimulation was also observed. CONCLUSION: In conclusion ginger extract inhibits macrophage activation and APC function and indirectly inhibits T cell activation.

Effect of 6-gingerol on pro-inflammatory cytokine production and costimulatory molecule expression in murine peritoneal macrophages.

BACKGROUND: Pro-inflammatory cytokines produced primarily by macrophages are key elements in many surgical conditions including sepsis, ischemia-reperfusion injury, and transplant rejection. Herbal products are being used as alternative treatments in such inflammatory conditions. Ginger is known for its ethno-botanical applications as an anti-inflammatory agent. 6-gingerol is one of the active ingredients of ginger that imparts ginger with its anti-inflammatory properties. We hypothesized that the anti-inflammatory effect of 6-gingerol is because of inhibition of macrophage activation, more specifically by an inhibition of pro-inflammatory cytokines and antigen presentation by lipopolysaccharide (LPS) activated macrophages. METHODS: To study the effect of 6-gingerol on pro-inflammatory cytokines, we measured the liberation of TNF-alpha, IL-1beta, and IL-12 by murine peritoneal macrophages exposed to several doses of 6-gingerol in the presence of LPS stimulation. We also studied the effect of 6-gingerol on the cell surface expression of B7.1, B7.2, and MHC II. Finally, we examined the APC function of the 6-gingerol treated macrophages by a primary mixed lymphocyte reaction. RESULTS: 6-gingerol inhibited the production of pro-inflammatory cytokines from LPS stimulated macrophages but had no effect on the LPS-induced expression of B7.1, B7.2, and MHC II. The APC function of LPS stimulated macrophages was also unaffected by 6-gingerol treatment. CONCLUSION: Our data indicate that 6-gingerol selectively inhibits production of pro-inflammatory cytokines from macrophages but does not affect either the APC function or cell surface expression of MHC II and costimulatory molecules. We, thus, provide a mechanistic insight into the anti-inflammatory properties of 6-gingerol that may be useful to treat inflammation without interfering with the antigen presenting function of macrophages.

A novel extracorporeal kidney perfusion system: a concept model.

The number of patients awaiting kidney transplantation has more than doubled in the past decade while the number of available donor organs has seen only a modest increase, leading to a critical shortage of organs. In response to this extreme shortage, the criteria for accepting organs have been modified to include marginal donors such as non-heart beating donors (NHBD). In these kidneys, determining viability is important for success of transplantation. Therefore, a study was undertaken to develop a system that would allow the extracorporeal assessment of function and compatibility of the donor organ before the patient is exposed to the risks associated with surgery. Following bilateral nephrectomy, the kidneys of 10 pigs (approximately 30 kg) were connected to a commercially available hypothermic pulsatile kidney perfusion apparatus. This system was modified to allow for normothermic pulsatile renal perfusion using the potential recipient's blood, via vascular access. These kidneys were perfused with the animal's blood for a minimum of two hours while various parameters were monitored. Perfusion pressures were kept between 60 and 90 mmHg, which correlated to flows between 70 and 150 mL/min. A decrease in perfusion pressure with a concomitant rise in flow over the two-hour period served as a good predictor of a viable and compatible graft. The modified kidney preservation system allows the normothermic, pulsatile extracorporeal perfusion of donor kidneys with the ability to monitor resistance to flow and urine production. This model also allows observation of the kidney for signs of hyperacute rejection. Further research needs to be conducted in order to determine if the system represents a methodology to increase the pool of available donor organs.

The effect of allo-injury in an in vitro model of allograft microvasculature.

Endothelial cells are critical to the integrity of allograft vasculature and can be damaged by alloreactive T cells or soluble mediators of alloreactivity. The biochemical effects of T cell-mediated damage to the endothelial cells have been studied, but not the structural and morphological effects of allo-injury on endothelial cells in the allograft. We utilized an assay that reproduces microvasculature in vitro to study the effect of alloreactivity on endothelial cells. In this assay, endothelial cells are induced into capillary-like networks that simulate microvascular capillaries. We studied the effect of allogeneic T cells and of soluble mediators from both mixed lymphocyte cultures (MLCs) and rejected heart allograft tissue on the in vitro capillaries. We found that both allogeneic T cells and soluble mediators inhibit the formation of the in vitro endothelial capillaries, suggesting that they cause a mild-to-moderate dysfunction of the endothelial cells. The inhibitory effect of the soluble mediators seems to be mediated, at least partly, by IFN-gamma, since this effect was prevented by antibody to IFN-gamma. Furthermore, pre-incubation of the in vitro capillaries with IFN-gamma appeared to magnify the effect of allogeneic T cells, as shown by a complete breakdown of well-formed in vitro capillary networks. Our experiments suggest that the in vitro capillary-tube model reflects structural injury to allograft vasculature by alloreactive T cells and their soluble mediators.

Cross-species comparison of gene expression between human and porcine tissue, using single microarray platform--preliminary results.

INTRODUCTION: Xenotransplantation is a potential solution for inadequate supply of donor organs. Pigs are considered the ideal donor for kidney transplantation to human recipients, therefore it is important to understand the gene regulation in the porcine organs. Oligonucleotide array technology has been utilized largely for human, mouse and rat gene expression studies only. Its use with porcine genes has not been reported. We investigated the possibility of studying gene regulation in porcine kidney with a human GeneChip microarray platform. METHODS: To assess the feasibility of using a single microarrray platform for comparison of expressing data across different species (human and pig), we compared the gene expression profiles of human brain, human kidney and pig kidney using the Affymetrix U-133 A human GeneChip, which contains probes for 22,283 genes. Kidney biopsies from pigs and humans, with normal histology, were used to obtain RNA for porcine and human samples, while a commercially available adult whole cortex total RNA sample (Clontech) was used for the human sample. We assessed the intensity ratio for housekeeping and tissue specific genes. To examine the potential for non-specific binding to create false positive errors in our data, we compared the expression profiles in our experiments to a number of public databases. RESULTS: There were approximately the same number of genes expressed at higher levels in the pig kidney as in the human kidney and human brain. The major differences in gene expression were found for genes with tissue specific patterns of expression. Eighty genes were increased in human brain vs. human and pig kidney samples. Two hundred and eighty genes were increased in human and pig kidney vs. human brain samples. Of the top 25 genes increased in pig kidney compared with human brain, we were able to cross-reference 18 genes to the Unigene and SAGE public databases. We confirmed the expected higher levels of expression in the kidney in 18 genes. Of the top 25 genes increased in human brain vs. pig kidney, we were able to cross-reference 20 genes to the Unigene and SAGE databases and confirm the expected higher expression levels in brain in 17 genes with three inconclusive genes. CONCLUSION: This low level of false positive findings, at this preliminary stage, supports the concept of using human GeneChip microarray platform to compare gene expression profiles between pig and human tissues in the absence of a porcine microarray platform. Our study opens a new avenue into the analysis of porcine genes relevant to xenotransplantation.

Signal transduction pathway in endothelial dysfunction.

BACKGROUND: Endothelial dysfunction is an important feature of sepsis, acute respiratory distress syndrome (ARDS), and other infectious conditions. Previously, we reported an in vitro model to study endothelial dysfunction, in which endothelial cells are induced to form capillary tube networks by culturing on a basement membrane matrix (Matrigel). In this study, we defined the signal transduction pathways that lead to endothelial cell function and capillary disruption characteristic of sepsis and other infectious conditions. METHODS: Human aortic endothelial cells (HAEC) were cultured on a laminin-rich matrix to form capillary-like networks. The HAECs were treated with a protein tyrosine phosphatase inhibitor (sodium orthovanadate), a phosphoinositon-3-phosphate inhibitor (wortmannin), or a protein kinase C inhibitor (bisindolylmaleimide) before capillary tubes had formed or after the capillary tubes had matured. The degree of capillary tube formation was quantified by counting the intersection of capillary networks in triplicate wells. Statistical significance was determined by analysis of variance. RESULTS: Endothelial dysfunction occurred after inhibition of protein tyrosine phosphatase or protein kinase C. Whereas inhibition of phosphoinositon-3-phosphate did not cause endothelial dysfunction, sodium orthovanadate (2-20 microM) and bisindolylmaleimide (2-10 microM) significantly reduced capillary networks. The mean +/- SD of the number of capillary tubes in the control, sodium orthovanadate-treated, and bisindolylmaleimide-treated groups were 251.0 +/- 7.0, 65.6 +/- 9.9 (p < 0.001), and 181.7 +/- 0.1 (p < 0.001), respectively. Sodium orthovanadate (20-200 microM) and bisindolylmaleimide (10-100 microM) inhibited capillary tube formation. At higher concentrations, sodium orthovanadate (> 200 microM) and bisindolylmaleimide (>100 microM) disrupted mature capillary tubes. CONCLUSIONS: Our results suggest that PKC and protein tyrosine phosphatase play a role in endothelial dysfunction by interfering with the phosphorylation signals within endothelial cells. These mechanisms may be important in the endothelial dysfunction in sepsis and other infectious conditions.

Augmentation of ischemia/reperfusion injury to endothelial cells by cyclosporin A.

Ischemia/reperfusion (I/R) carries significant injury to endothelial cells in transplanted organs and is an important factor in chronic rejection. Immunosuppressive drugs, notably cyclosporin A (CyA) and FK506, can potentially augment this injury. Here, our goal was to determine the combined effects of I/R and CyA or FK506 on endothelial cells. Transformed mouse endothelial cells (SVEC 4-10) were subjected to ischemia or I/R for 2-24 hours by incubating cells in 100 per cent N2 (ischemia) followed by 5 per cent CO2 and 95 per cent O2 (reperfusion) for 24 hours. In separate experiments, CyA or FK506 was added to cells subjected to ischemia or I/R. Nonviable cells were determined by Trypan blue exclusion assay. All experiments (done in triplicate) were analyzed by Student's t test. Increasing ischemia times resulted in a greater number of nonviable cells (2% nonviable cells at 0 hours and 57% at 24 hours of I/R). Addition of CyA significantly increased the number of nonviable cells when compared with the control (I/R only) group (P = 0.014). Interestingly, FK506 did not increase the percentage of nonviable cells compared with the control group (P = 0.2). Unlike FK506, CyA augments I/R injury to endothelial cells in vitro. These findings could be relevant in chronic rejection and transplantation.

Initial angiogenic response in reduced renal mass after transplantation.

INTRODUCTION: Shortage of organs is a major problem in kidney transplantation and requires novel strategies to increase the number of kidney transplants. To reduce the shortage of kidneys, we have proposed transplantation of two halves of one kidney into two recipients (hemirenal transplantation, HRT) and have shown its feasibility in pig and human kidneys. However, reduced renal mass can lead to progressive renal failure in rodents and can reduce the longevity of kidney transplants in humans. Recent studies suggest that derangement of angiogenesis plays a role in the progressive renal failure after reduction in renal mass in rodents. However, since the renal physiology of rats is different from that of large animals, we studied angiogenesis in reduced renal mass transplants in pigs and determined if the reduction in renal mass has the same effect in large animals as that in rodents. MATERIALS AND METHODS: Kidney autotransplantation was performed in domestic outbred swine. Heminephrectomy of the autotransplanted kidney and nephrectomy of the contralateral kidney were performed 1 week after transplantation to reduce the renal mass. Four weeks after transplantation, the pigs were sacrificed and the hemirenal and control nephrectomy specimens were processed for morphometric analysis of glomerular capillary density and immunohistochemical analysis of VEGF expression. Soluble extracts from the kidneys were tested in an in vitro angiogenesis assay to determine their activity to influence angiogenesis. Statistical analysis with ANOVA was performed on the glomerular capillary density in kidney specimens. RESULTS: All these parameters of angiogenesis were increased in the reduced renal mass autotransplants as compared to normal kidneys or whole kidney autotransplants. Glomerular capillary density was increased significantly after reduction in renal mass. VEGF expression also was increased progressively by the third week after reduction in renal mass. Soluble extract from the reduced renal mass transplants significantly increased the in vitro angiogenesis. CONCLUSION: This is the first study to demonstrate that angiogenesis is increased in the initial stages of reduction in renal mass after transplantation in a large animal model. Increased angiogenesis was found in this model earlier than reported in small animal models (2 weeks in pigs versus 6 weeks in rats). Taken together with other studies, our data suggest that derangement in angiogenesis could play an important role in long-term graft function after hemirenal transplantation.

beta1-integrin-ligand disengagement induces in vitro capillary tube disruption mediated by p38 MAPK activity.

BACKGROUND: Disrupting cell-matrix interactions may lead to capillary injury as seen in sepsis and transplant rejection. Previously, we demonstrated capillary disruption mediated by beta1-integrin-ligand disengagement. We now determine whether p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) pathways are involved in this capillary injury. METHODS: Endothelial capillaries on Matrigel were preincubated with a p38 MAPK inhibitor (SB203580), ERK pathway inhibitor (PD98059), or dimethyl sulfoxide. Subsequently, a beta1-integrin blocking (P5D2) or an irrelevant antibody was added. After 24 hours, capillary integrity was quantified as capillary intersections/well. Antibody-treated cell lysates then were immunoprecipitated with either a phospho-p38 MAPK or phospho-ERK1/2 antibody. Kinase activity was measured with ATF-2 and Elk-1 fusion proteins as substrates for p38 MAPK and ERK, respectively, followed by Western blotting. RESULTS: P5D2 disrupted capillary tubes. Increased p38 MAPK activity at 8 hours and ERK activity at 2 and 8 hours were seen in P5D2-treated lysates. Preincubation with SB203580, but not with PD98059 or DSMO, significantly reduced capillary tube disruption. CONCLUSIONS: The beta1-integrin-ligand disengagement resulted in capillary disruption and stimulated p38 MAPK and ERK activity. In spite of activation of both pathways, the p38 MAPK but not the ERK pathway inhibitor prevented beta1-integrin antibody effects. Inhibiting p38 MAPK may mitigate capillary injury associated with sepsis and transplant rejection.

Amelioration of cyclosporin A effect on microvasculature by endothelin inhibitor.

BACKGROUND: We have previously shown that endothelial injury by cyclosporin A (CyA) is associated with an increased endothelin-1 (ET-1) release. We now sought to determine, in an animal model of angiogenesis, if inhibiting the effect of ET-1 on endothelial cells (ECs) would reverse the CyA-mediated endothelial injury in an animal model of angiogenesis. METHODS: An angiogenic mixture of Matrigel (0.5 ml), fibroblast growth factor (1 ng/ml), vascular endothelial growth factor (100 ng/ml), and heparin (64 unit/ml) was injected as a subcutaneous plug in the flank of C3H mice (n = 5). In experimental groups CyA (20 mg/ml), CyA, and BQ 123 (ET-A receptor antagonist), CyA and PD 142893 (ET-A and ET-B receptor antagonist), or CyA and ET-1 antibody were added to the angiogenic mixture. Angiogenesis in the mixture was quantified by modified planimetric point counting method in skin/Matrigel cross-sections stained with factor VIII to highlight endothelial neocapillaries. Mean +/- SD of angiogenic area was analyzed with analysis of variance and Bonferroni test. The survival curves obtained by Kaplan-Meier analysis were compared between the groups, and the statistical significance of survival and mortality rates was computed by log rank's and Fisher's exact test, respectively. RESULTS: The mean +/- SD of angiogenic area in control animals (without CyA in the angiogenic mixture) was 56.76 +/- 4.2. CyA inhibited angiogenesis in the subcutaneous angiogenic plug. Adding CyA to the angiogenic mixture significantly reduced angiogenic area (5.33 +/- 1.4, P <.001) while vehicle for CyA had no such effect (56.33 +/- 3.8, P =.10). Polyclonal ET-1 antibody or PD 142893 ameliorated the effect of CyA, whereas BQ 123 did not. The mean angiogenic areas in animals with ET-1 antibody, PD 142893, or BQ 123 in the angiogenic mixture were 57.20 +/- 7.5 (P =.06), 46.00 +/- 11.5 (P = 1.0), 8.60 +/- 2.9 (P <.001), respectively. CONCLUSIONS: Our data show that blocking ET-B receptors specifically ameliorates the microvascular injury to the neocapillaries in angiogenesis caused by CyA. Antiendothelin-1 antibody and ETR antagonist (PD 142893) could, therefore, reduce the ill effects of CyA on microvascular endothelium.

Morphological and biochemical effects of immunosuppressive drugs in a capillary tube assay for endothelial dysfunction.

Immunosuppressive drugs common in clinical transplantation are known to have untoward effects on the vascular system. The effects of some drugs, notably cyclosporin A (CyA), have been studied on the vascular system, while those of others have not. In the vascular system, endothelial cells are the predominant cell type exposed to intravascular concentrations of immunosuppressive drugs. We therefore studied the effects of drugs common in clinical transplantation on endothelial cells in a capillary tube assay. The endothelial cells in the capillary tubes are morphologically more similar to those in the microvasculature than endothelial cells in monolayers. We studied the kinetics and extent of capillary tube formation and prostacyclin (PGI2) and endothelin-1 (ET-1) release from the in vitro capillaries to determine the morphological and biochemical effects of five immunosuppressive agents on endothelial function. We found a significant difference in the morphological and biochemical effects of the two common calcineurin inhibitors, CyA and tacrolimus (FK506) on capillary morphology in vitro. The former had a pronounced injurious effect on the morphology of the in vitro capillaries, while the latter did not. CyA also significantly increased ET-1 release by the capillaries, but FK506 did not. Mycophenolate mofetil (MMF) was the only other agent that had a moderately injurious effect on the morphology of the in vitro capillaries. Sirolimus (rapamycin) and dexamethasone, similar to FK506, had no effect on the capillary morphology. All these agents, except dexamethasone, increased PGI2 release. Our data suggest that CyA adversely affects the morphology of the microvasculature and that this is mediated, at least partly, by an increased ET-1 release by endothelial cells exposed to CyA. These findings describe a novel effect of CyA and MMF on endothelial cells that could be relevant to understanding the mechanisms of immunosuppressive drug-mediated endothelial injury in clinical transplantation.

Role of endothelin-1 in microvascular dysfunction caused by cyclosporin A.

BACKGROUND: Endothelin-1 (ET-1), a potent vasoconstrictive peptide, is implicated in cyclosporin A (CyA) vasculopathy. Previously we have demonstrated, in an in vitro model of endothelial capillaries, that CyA inhibits the formation of the capillaries and, in high doses, disrupts the capillaries. This study addresses the role of ET-1 in CyA-induced endothelial dysfunction of the in vitro capillaries. STUDY DESIGN: Endothelial cells (ECs) were cultured on a laminin-rich matrix, Matrigel, to form capillary-like networks. The ECs were treated with CyA either before capillary tube formation or after capillary tubes had formed. ppET-1 gene expression was studied by reverse transcriptase polymerase chain reaction. To determine if ET-1 was involved in the CyA-mediated disruption of the in vitro capillaries, ET-1 binding to the endothelial cells was blocked by ET-1 antibody and ET receptor antagonists. The effects of exogenous ET-1 were also studied. The results were quantified by counting the number of capillary networks, and the statistical significance was determined with ANOVA. RESULTS: ppET-1 was expressed in ECs during capillary tube formation, but disappeared once capillary tubes had matured. The ppET-1 gene expression reappeared when the capillary tubes were exposed to CyA. Exogenous ET-1 partially reversed the inhibition of tube formation by cyclohexamide, allowing initiation of tube formation. CyA-mediated capillary dysfunction was completely prevented by an anti-ET-1 antibody and an ET-B receptor antagonist. CONCLUSIONS: Endothelin-1 plays a significant role in CyA-induced endothelial dysfunction and may play a role in allograft vasculopathy. Blocking of ET-1 is a strategy to prevent endothelial dysfunction caused by CyA.