Matrix metalloproteinase induction in post-transplant obliterative bronchiolitis.

BACKGROUND: Epithelial cell injury, inflammation, fibrosis, and airway obliteration are associated in post-transplant obliterative bronchiolitis. Fibrosis is a consequence of fibroblastic activity and of collagen deposition after disturbances in the balance of protein formation and degradation. Proteolytic enzymes such as the matrix metalloproteinases mediate degradation. To assess matrix metalloproteinases during obliterative bronchiolitis development, we studied porcine, heterotopic bronchial allografts. METHODS: A total of 119 allografts or autografts were harvested serially at 3 to 60 days after transplantation and processed for histology and in situ hybridization for matrix metalloproteinases 2 and 9. Immunocytochemistry for vimentin and alpha-smooth-muscle-cell actin was performed with specific antibodies. RESULTS: Implants had initial ischemic injury to airway epithelium and to the bronchial wall. Recovery was rapid in autografts and in immunosuppressed allografts. In matrix metalloproteinase-2 mRNA activity in fibroblasts, correlation with endothelial expression and expression in macrophages occurred during intense fibroproliferation. We observed intense matrix metalloproteinase-9 positivity during onset of inflammation and fibroproliferation in endothelial cells (p < 0.01), fibroblasts (p < 0.05), macrophages (p < 0.05), and lymphocytes (p < 0.05). Matrix metalloproteinase-9 mRNA activity in fibroblasts correlated with that in endothelial and inflammatory cells and also proved predictive of early obliteration. CONCLUSIONS: Matrix metalloproteinase-2, and especially matrix metalloproteinase-9, gene activity was associated with onset of inflammation and fibroblastic proliferation in allografts, predicting early obliteration. Although this may be the case in the model described, its role in human-allograft post-transplant obliterative bronchiolitis requires further supportive data.

Tumor necrosis factor-alpha in a porcine bronchial model of obliterative bronchiolitis.

BACKGROUND: In posttransplant obliterative bronchiolitis (OB), the major pathologic features are inflammation, epithelial cell injury, fibrosis, and obliteration of the small airways. Tumor necrosis factor (TNF)-alpha is a cytokine known to mediate and augment the inflammatory reaction and to enhance fibroblast proliferation. We assessed the role of TNF-alpha in the development of OB in our heterotopic porcine bronchial transplantation model. METHODS: Three groups were formed: autografts, nontreated allografts, and allografts treated with preoperative anti-TNF-alpha monoclonal antibody (infliximab) infusion. The implants were harvested on days 2, 4, 7, 11, 14, 21, and 28 for histologic and immunohistochemical analysis. RESULTS: TNF-alpha inhibition reduced inflammation, rate of epithelial loss, fibrosis, and obliteration early in the development of OB. In the epithelium, the numbers of TNF-alpha-positive epithelial and inflammatory cells and macrophages were significantly lower in treated than in nontreated allografts on day 4; furthermore, in the epithelium and in the bronchial wall, invasion of CD8+ lymphocytes was significantly decreased during the first week. CONCLUSIONS: These results indicate that TNF-alpha promotes the development of OB, and inhibition of TNF-alpha may prove beneficial in a clinical setting.

Nitric oxide in the development of obliterative bronchiolitis in a heterotopic pig model.

BACKGROUND: Inflammation, epithelial cell injury, and development of fibrosis and airway obliteration are the major histological features of posttransplant obliterative bronchiolitis (OB). The expression of inducible nitric oxide synthase (iNOS) in the damaged epithelium, accompanied by peroxynitrite, suggests that endogenous nitric oxide (NO) mediates the epithelial destruction preceding obliteration. To elucidate the role of NO in this cascade, heterotopic bronchial allografts were studied in pigs. METHODS: Allografts or autografts were harvested serially 3-90 days after transplantation and processed for histology and immunocytochemistry for iNOS, nitrotyrosine, a marker of peroxynitrite formation, and superoxide dismutase (SOD). RESULTS: During initial ischemic damage to the epithelium, iNOS, nitrotyrosine, and SOD were found to be strongly expressed in the epithelium of all implants as well as later, after partial recovery, parallel to onset of epithelial destruction and subsequent airway obliteration in allografts. The levels of expression of iNOS in fibroblasts during the early phase of obliteration paralleled the onset of fibrosis. Constant expression of iNOS and SOD, but not nitrotyrosine, occurred in autografts and allografts with blocked alloimmune response. CONCLUSIONS: These findings suggest that an excessive amount of NO promotes posttransplant obliterative bronchiolitis by destroying airway epithelium and stimulating fibroblast activity. SOD may provide protection by binding reactive molecules and preventing peroxynitrite formation.

Platelet deposition on stainless steel, spiral, and braided polylactide stents. A comparative study.

Platelets play a key role in (sub)acute thrombotic occlusion after stenting. We examined the possible differences between biodegradable polylactide (PLA) and stainless steel (SS) stents in platelet attachment and morphology after whole blood perfusion. PLA stents of different configurations (spiral/braided) and polycaprolactone-polylactide (PCL-PLA)-coatings, or SS stents were implanted into a PVC tube (Ø 3.2 mm), with or without precoating of the tube with type-I collagen. PPACK (30 microM)-anticoagulated blood with (3)H-serotonin prelabeled platelets was perfused (flow rate: 30 ml/min, 90 s) over the stents. Platelet deposition was assessed by scintillation counting and morphology by scanning electron microscopy (SEM). To examine coagulation activation, plasma prothrombin fragments (F1 + 2) were measured before and after the perfusion. Protein deposition on PLA/SS stents was assessed at augmented shear forces mimicking coronary flow (rate: 60 ml/min, 60 s) under minimal anticoagulation (PPACK 1 microM). More platelets deposited on PLA stents than on SS stents under all study conditions (p < 0.03). Under anticoagulation (PPACK 30 microM) the generation of F1 + 2 remained unaltered. Under higher flow rate and limited anticoagulation SS stents accumulated 3.27 +/- 0.75 microg and PLA stents 5.25 +/- 1.74 microg of protein (Mean +/- SD, p <0.95). Among all biodegradable stents, the braided PLA stent coated with PCL-PLA-heparin accumulated the fewest platelets (p < 0.02). In SEM, signs of platelet activation on braided heparin-coated PLA stents, when compared with uncoated braided PLA/SS stents, appeared modest. In conclusion, PCL-PLAheparin coating of biodegradable stents may enhance their hemocompatibility, expressed by less platelet deposition. Nevertheless, materials, design, and coating techniques of biodegradable stents must be further developed.

Epithelial apoptosis in experimental obliterative airway disease after lung transplantation.

BACKGROUND: Epithelial damage is an important feature in the pathogenesis of obliterative airway disease. We investigated the extent of epithelial apoptosis in this process in pig bronchial allografts. METHODS: The bronchial grafts (total, n = 200) were placed subcutaneously into recipients. Three allograft groups were formed: the first group had no immunosuppression therapy; the second received triple therapy with 10 mg/kg/day cyclosporine, 2 mg/kg/day azathioprine, and 20 mg/day methylprednisolone; and the third was given triple therapy in which azathioprine was replaced with 1.5 mg/kg/day everolimus (40-O-[2-hydroxyethyl]-rapamycin). The fourth group, which had allograft and autograft implants, received only 1.5 mg/kg/day everolimus. The implants were serially removed during 3 months of follow-up. We evaluated graft histology and analyzed the apoptotic index percentage (apoptotic cells / total number of cells) of the bronchial epithelium using in situ 3'-end labeling of apoptotic DNA. RESULTS: Epithelial destruction and subsequent obliteration of the bronchial lumen were complete by Day 28 in non-treated allografts and in most allografts with inadequate immunosuppression to prevent these changes (those treated with cyclosporine, azathioprine, and methylprednisolone and those treated with everolimus only). The apoptotic indexes of the epithelium were high (>1% of the cells were apoptotic) and increased with concomitant epithelial destruction. In allografts with adequate immunosuppression to prevent epithelial destruction (those treated with cyclosporine, everolimus, and methylprednisolone) and in autografts, after initial damage, well-pre-served epithelium was maintained with low apoptotic indexes (<1% of the cells apoptotic). CONCLUSIONS: Apoptotic activity increased with progressing epithelial damage preceding bronchial obliteration. Our results give further evidence that apoptotic death of epithelial cells is an important mechanism in events that lead to graft deterioration in obliterative bronchiolitis after lung transplantation.

Platelet responses and coagulation activation on polylactide and heparin-polycaprolactone-L-lactide-coated polylactide stent struts.

Despite modern stent technology and effective antiplatelet therapy, metallic stents carry the risk of (sub)acute thrombosis. Our aim was to examine short-term differences in platelet deposition and coagulation activation between biodegradable polylactide (PLA), heparin-polycaprolactone-L-lactide-coated polylactide (hepa-P(CL95/L-LA5)-PLA), and stainless steel (SS) stent struts. Gel-filtered platelets (GFP) and platelet-rich plasma (PRP) were labeled with 10 nM (3)H-serotonin. Platelet deposition was measured after incubation of the stent struts in human serum albumin-coated wells at 37 degrees C in either GFP or PRP. Platelet morphology was studied by scanning electron microscopy (SEM). For coagulation activation, the stent struts were incubated in either PRP or platelet-poor plasma (PPP), anticoagulated with D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK), followed by measurement of fibrinogen, thrombin time (TT), prothrombin fragment 1+2 (F1+2), and thrombin-antithrombin complex (TAT). SS showed adherence of larger amounts of GFPs than did PLA at a platelet density of 300 x 10(6)/mL (p < 0.05). Furthermore, representative SEM studies showed more platelet spreading on SS than on PLA stent struts. Between PLA and SS, coagulation activity did not differ at any assessment. Based on prolonged TT values in plasma, the heparin coating strongly inhibited coagulation (p < 0.05). The values of soluble TAT and F1+2 for PLA were similar to those of controls, i.e., to incubated suspensions without a stent strut. In conclusion, when compared with stainless steel, both PLA and hepa-P(CL95/L-LA5)-PLA appear hemocompatible as intravascular stent materials.

Association between gastrointestinal symptoms and health-related quality of life after heart transplantation.

BACKGROUND: Heart transplantation (HTx) ameliorates the self-perceived health-related quality of life (HRQoL) of patients with terminal heart disease; gastrointestinal (GI) symptoms, due to obligatory immunosuppression, may contribute to impaired HRQoL post-HTx. METHODS: In this cross-sectional, exploratory study we aimed to investigate association between GI symptoms and HRQoL. The study consisted of 167 patients who had undergone HTx between 1985 and 2006 in Finland. Short-Form 36 (SF-36) Quality of Life and Gastrointestinal Symptom Rating Scale (GSRS) questionnaires were sent to the patients at the end of 2006 for capturing data on the HRQoL of HTx recipients. RESULTS: Higher or equal SF-36 scores describing the HRQoL dimensions compared with the Finnish age- and gender-matched reference population were as follows: physical functioning, 60.5%; role-physical, 67.5%; bodily pain, 62.6%; general health, 64.0%; vitality, 68.1%; social functioning, 68.1%; role-emotional, 70.0%; and mental health, 70.4%. The prevalence of troublesome GI symptoms (GSRS score >1) per GSRS dimension was 53.9% for diarrhea, 91.0% for indigestion, 60.6% for constipation, 73.4% for abdominal pain, 46.4% for reflux and 95.8% for any GI symptom. Diabetes contributed to the presence of diarrhea (odds ratio [OR]: 3.00; 95% confidence interval [CI]: 1.12 to 8.00), use of prednisolone to indigestion (OR: 3.21; 95% CI: 1.05 to 9.79) and increased age to constipation (OR: 1.04; 95% CI: 1.02 to 1.07). CONCLUSIONS: HRQoL after HTx is relatively good and comparable to the age- and gender-matched reference population. HRQoL is vulnerable to side-effects caused by the obligatory post-HTx immunosuppressive regimen, where GI symptoms play a major but clearly an underestimated role.

Magnetic resonance evaluation of luminal patency after polylactide stent implantation: an experimental study in a rabbit aorta model.

Intravascular metallic stents cause magnetic field distortions, disturbing luminal detection. Our aim was to evaluate the effect of polylactic acid (PLA) stents on magnetic resonance angiography (MRA). Biodegradable double spiral helical PLA stents ( n=12) or stainless steel (SS) ( n=6) stents were implanted into the infrarenal aortas of New Zealand White rabbits. All SS- and 6 PLA-stented animals as well as 6 non-operated control rabbits were imaged with gadolinium-enhanced MRA (1.5 T), and infrarenal aortic diameters (proximal, distal, and narrowest), together with the stent artifact, were measured. Six of the PLA-stented rabbits were followed up, and MRA was assessed at 2, 6, 9, and 12 months after the stent implantation. Image artifacts caused by the SS stents were visualized in all cases. The PLA stents caused no magnetic field distortion, allowing imaging of the underlying vessel including the vessel lumen. In the follow-up group of 6 rabbits with a PLA stent, 5 of 6 were patent at the end of the follow-up of 12 months. These stents allowed luminal detection at all time points, with no significant differences in aortic measurements emerging during the whole follow-up period. When immediately postoperatively scanned SS and PLA rabbits were compared with controls, no differences were observable in proximal diameters. Instead, the percentage of distal luminal loss was greater in the PLA-stented rabbits, as compared with SS stents ( p<0.01). The PLA stenting in small vessels allows evaluation of luminal patency with MRA both immediately after implantation and during follow-up.