Age-determined severity of anti-myeloperoxidase autoantibody-mediated glomerulonephritis in mice.

Background: Anti-neutrophil cytoplasmic antibody associated vasculitis (AAV) is a typical disease of the elderly. In AAV, there is an age-specific increase in disease incidence with age being a predictor of disease outcome. In this study, we aimed to determine the contribution of age to the development of AAV employing a mouse model of anti-myeloperoxidase (MPO) antibody-mediated glomerulonephritis. Methods: Anti-MPO IgG and lipopolysaccharide (LPS)-mediated glomerulonephritis was induced in 3- and 18-month-old C57Bl6 mice. Clinical and pathological parameters of disease severity, alterations in the immune system and kidney specific changes in these mice were evaluated. Results: Eighteen-month-old mice developed increased disease severity upon injection of anti-MPO IgG/LPS compared with 3-month-old mice. This was evidenced by increased albuminuria, more extensive glomerular capillary necrosis and increased glomerular neutrophil accumulation. Glomerular crescent formation was mild in both young and old mice. Old mice displayed higher plasma interleukin-6 levels as well as higher proportions of circulating neutrophils and activated monocytes compared with young mice. In addition, renal mRNA levels of inflammatory genes and endothelial adhesion molecules were higher in 18-month-old mice compared with 3-month-old mice. Conclusion: In conclusion, our results indicate that aged mice develop more severe clinical and pathological disease upon induction of anti-MPO IgG/LPS-mediated glomerulonephritis. These findings may be attributed to age-related changes in the immune system as well as in the kidney itself.

Treatment with Anti-HMGB1 Monoclonal Antibody Does Not Affect Lupus Nephritis in MRL/lpr Mice.

High mobility group box 1 (HMGB1) is a nuclear DNA binding protein that acts as an alarmin when secreted. HMGB1 is increased in systemic lupus erythematosus and might represent a potential therapeutic target. We investigated whether treatment with an anti-HMGB1 antibody affects the development of lupus nephritis in MRL/lpr mice. Seven-week-old MRL/lpr mice were injected intraperitoneally twice weekly for 10 wks with 50 µg monoclonal anti-HMGB1 (2G7, mouse IgG2b) (n = 12) or control antibody (n = 11). Control MRL/MPJ mice (n = 10) were left untreated. Every 2 wks, blood was drawn and urine was collected at wk 7, 11 and 17. Mice were sacrificed at 17 wks for complete disease evaluation. Plasma HMGB1 and anti-HMGB1 levels were increased in MRL/lpr mice compared with control MRL/MPJ mice. There were no differences in albuminuria, urine HMGB1 and plasma levels of complement C3, anti-dsDNA and proinflammatory cytokines between untreated and treated mice at any time point. Lupus nephritis of mice treated with anti-HMGB1 monoclonal antibody (mAb) was classified as class III (n = 3) and class IV (n = 9), while mice treated with control mAb were classified as class II (n = 4), class III (n = 2) and class IV (n = 5). IgG and C3 deposits in kidneys were similar in mice treated with anti-HMGB1 mAb or control mAb. In conclusion, treatment with monoclonal anti-HMGB-1 antibody 2G7 does not affect development of lupus nephritis, disease progression or proinflammatory cytokine levels in MRL/lpr mice. This result indicates that blocking of HMGB1 by this neutralizing antibody does not affect lupus nephritis in MRL/lpr mice.

Microsphere-Based Rapamycin Delivery, Systemic Versus Local Administration in a Rat Model of Renal Ischemia/Reperfusion Injury.

PURPOSE: The increasing prevalence and treatment costs of kidney diseases call for innovative therapeutic strategies that prevent disease progression at an early stage. We studied a novel method of subcapsular injection of monodisperse microspheres, to use as a local delivery system of drugs to the kidney. METHODS: We generated placebo- and rapamycin monodisperse microspheres to investigate subcapsular delivery of drugs. Using a rat model of acute kidney injury, subcapsular injection of placebo and rapamycin monodisperse microspheres (monospheres) was compared to subcutaneous injection, mimicking systemic administration. RESULTS: We did not find any adverse effects related to the delivery method. Irrespective of the injection site, a similar low dose of rapamycin was present in the circulation. However, only local intrarenal delivery of rapamycin from monospheres led to decreased macrophage infiltration and a significantly lower amount of myofibroblasts in the kidney, where systemic administration did not. Local delivery of rapamycin did cause a transient increase in the deposition of collagen I, but not of collagen III. CONCLUSIONS: We conclude that therapeutic effects can be increased when rapamycin is delivered subcapsularly by monospheres, which, combined with low systemic concentrations, may lead to an effective intrarenal delivery method.

Flush at room temperature followed by storage on ice creates the best lung graft preservation in rats.

Current clinical lung preservation techniques have not eliminated ischaemia-reperfusion (I/R) injury, despite many improvements. The optimal combination of flush and storage temperatures remain unclear in lung preservation. This is the first study to investigate a range of temperatures with 24-h inflated storage using consistent state-of-the-art preservation techniques. A rat lung transplant model was used to investigate the optimal combination of flush and storage temperatures. In six groups, rat lungs were flushed at 4 °C, 10 °C or room temperature (F(4) /F(10) /F(Rt)) with Perfadex and stored inflated for 24 h in Perfadex on melting ice or at 10 °C (S(ice) /S(10)). Left donor lungs were transplanted for analysis. During 2-h reperfusion, the lung graft function was measured (blood gases, maximum ventilation pressure and static compliance) and lung graft injury was also assessed (W/D ratio, total lung protein, Tryptase, Myeloperoxidase). Right donor lungs were assessed for W/D ratio only after flush and storage. For baseline measurements, left lungs without intervention were used. The combination of F(Rt) -S(ice) showed a significantly higher pO(2), lower P(max), low W/D ratios and total protein levels of left lungs after reperfusion when compared with F(4) -S(ice) and baseline. Storage at 10 °C did not improve preservation. We conclude that F(Rt) -S(ice) creates the best lung graft preservation.

Effects of p38 mitogen-activated protein kinase inhibition on anti-neutrophil cytoplasmic autoantibody pathogenicity in vitro and in vivo.

OBJECTIVE: To determine whether inhibition of p38 mitogen-activated protein kinase (p38MAPK) reduces the pathogenicity of anti-neutrophil cytoplasmic autoantibodies (ANCAs) in vitro and in vivo. METHODS: The effects of the p38MAPK-specific inhibitor AR-447 were studied in vitro using neutrophil respiratory burst and degranulation assays, and in lipopolysaccharide (LPS)-stimulated human glomerular endothelial cells. In vivo, p38MAPK inhibition was investigated in a mouse anti-myeloperoxidase (MPO) IgG/LPS glomerulonephritis model. Mice were treated orally with AR-447 daily, starting before (pretreatment group) or 24 h after disease onset (treatment group), and killed after 1 or 7 day(s). RESULTS: In vitro, AR-447 diminished neutrophil respiratory burst and degranulation induced by patient-derived MPO-ANCA and proteinase 3 (Pr3)-ANCA. In glomerular endothelial cells, AR-447 reduced LPS-induced secretion of IL-6 and IL-8, but not of MCP-1. In mice, pretreatment with AR-447 reduced albuminuria 1 day after induction of glomerulonephritis. After 7 days, no effects on urinary abnormalities were observed upon AR-447 pretreatment or treatment. Also, glomerular neutrophil accumulation was not diminished. In contrast, glomerular macrophage accumulation and the formation of glomerular crescents was significantly reduced by AR-447 pretreatment (vehicle: 12.5 ± 5.6% crescentic glomeruli; AR-447: 7.7 ± 2.7%) and treatment (vehicle 14.6 ± 1.8%; AR-447 6.0 ± 3.4%) at 7 days. CONCLUSION: This study shows that p38MAPK inhibition markedly reduces ANCA-induced neutrophil activation in vitro. In vivo, p38MAPK inhibition partly reduced crescent formation when the drug was administered prior to disease induction and after disease onset, suggesting that besides p38MAPK activity other signalling pathways contribute to the disease activity.

IgG glycan hydrolysis attenuates ANCA-mediated glomerulonephritis.

Anti-neutrophil cytoplasmic autoantibodies (ANCA) directed against myeloperoxidase (MPO) and proteinase 3 (Pr3) are considered pathogenic in ANCA-associated necrotizing and crescentic glomerulonephritis (NCGN) and vasculitis. Modulation of ANCA IgG glycosylation may potentially reduce its pathogenicity by abolishing Fc receptor-mediated activation of leukocytes and complement. Here, we investigated whether IgG hydrolysis by the bacterial enzyme endoglycosidase S (EndoS) attenuates ANCA-mediated NCGN. In vitro, treatment of ANCA IgG with EndoS significantly attenuated ANCA-mediated neutrophil activation without affecting antigen-binding capacity. In a mouse model of anti-MPO IgG/LPS-induced NCGN, we induced disease with either unmodified or EndoS-treated (deglycosylated) anti-MPO IgG. In separate experiments, we administered EndoS systemically after disease induction with unmodified anti-MPO IgG. Pretreatment of anti-MPO IgG with EndoS reduced hematuria, leukocyturia, and albuminuria and attenuated both neutrophil influx and formation of glomerular crescents. After inducing disease with unmodified anti-MPO IgG, systemic treatment with EndoS reduced albuminuria and glomerular crescent formation when initiated after 3 but not 24 hours. In conclusion, IgG glycan hydrolysis by EndoS attenuates ANCA-induced neutrophil activation in vitro and prevents induction of anti-MPO IgG/LPS-mediated NCGN in vivo. Systemic treatment with EndoS early after disease induction attenuates the development of disease. Thus, modulation of IgG glycosylation is a promising strategy to interfere with ANCA-mediated inflammatory processes.

Methods for Experimental Allergen Immunotherapy: Subcutaneous and Sublingual Desensitization in Mouse Models of Allergic Asthma.

Allergic asthma is characterized by airway hyperresponsiveness, remodeling, and reversible airway obstruction. This is associated with an eosinophilic inflammation of the airways, caused by inhaled allergens such as house dust mite or grass pollen. The inhaled allergens trigger a type-2 inflammatory response with the involvement of innate lymphoid cells (ILC2) and Th2 cells, resulting in high immunoglobulin E (IgE) antibody production by B cells and mucus production by airway epithelial cells. As a consequence of the IgE production, subsequent allergen reexposure results in a classic allergic response with distinct early and late phases, both resulting in bronchoconstriction and shortness of breath. Allergen-specific immunotherapy (AIT) is the only treatment that is capable of modifying the immunological process underlying allergic responses including allergic asthma. Both subcutaneous AIT (SCIT) as well as sublingual AIT (SLIT) have shown clinical efficacy in long-term suppression of the allergic response. Although AIT treatments are very successful for rhinitis, application in asthma is hampered by variable efficacy, long duration of treatment, and risk of severe side effects. A more profound understanding of the mechanisms by which AIT induces tolerance to allergens in sensitized individuals is needed to be able to improve its efficacy. Mouse models have been very valuable in preclinical research for characterizing the mechanisms of desensitization in AIT and evaluating novel approaches to improve its efficacy. Here, we present a rapid and reproducible mouse model for allergen-specific immunotherapy. In this model, mice are sensitized with two injections of allergen adsorbed to aluminum hydroxide, followed by subcutaneous injections (SCIT) or sublingual administrations (SLIT) of allergen extracts as an immunotherapy treatment. Finally, mice are challenged by intranasal allergen administrations. We will also describe the protocols as well as the most important readout parameters for the measurements of invasive lung function, serum immunoglobulin levels, isolation of bronchoalveolar lavage fluid (BALF), and preparation of cytospin slides. Moreover, we describe how to perform ex vivo restimulation of lung single-cell suspensions with allergens, flow cytometry for identification of relevant immune cell populations, and ELISAs and Luminex assays for assessment of the cytokine concentrations in BALF and lung tissue.

High dose vitamin D3 empowers effects of subcutaneous immunotherapy in a grass pollen-driven mouse model of asthma.

Allergen-specific immunotherapy (AIT) has the potential to provide long-term protection against allergic diseases. However, efficacy of AIT is suboptimal, while application of high doses allergen has safety concerns. The use of adjuvants, like 1,25(OH)2VitD3 (VitD3), can improve efficacy of AIT. We have previously shown that low dose VitD3 can enhance suppression of airway inflammation, but not airway hyperresponsiveness in a grass pollen (GP)-subcutaneous immunotherapy (SCIT) mouse model of allergic asthma. We here aim to determine the optimal dose and formulation of VitD3 for the GP SCIT. GP-sensitized BALBc/ByJ mice received three SCIT injections of VitD3-GP (30, 100, and 300 ng or placebo). Separately, synthetic lipids, SAINT, was added to the VitD3-GP-SCIT formulation (300 nmol) and control groups. Subsequently, mice were challenged with intranasal GP, and airway hyperresponsiveness, GP-specific IgE, -IgG1, and -IgG2a, ear-swelling responses (ESR), eosinophils in broncho-alveolar lavage fluid and lung were measured. VitD3 supplementation of GP-SCIT dose-dependently induced significantly enhanced suppression of spIgE, inflammation and hyperresponsiveness, while neutralizing capacity was improved and ESR were reduced. Addition of VitD3 further decreased Th2 cytokine responses and innate cytokines to allergens in lung tissue by GP-SCIT. However, addition of synthetic lipids to the allergen/VitD3 mixes had no additional effect on VitD3-GP-SCIT. We find a clear, dose dependent effect of VitD3 on GP-SCIT-mediated suppression of allergic inflammation and airway hyperresponsiveness. In contrast, addition of synthetic lipids to the allergen/VitD3 mix had no therapeutic effect. These studies underscore the relevance of VitD3 as an adjuvant to improve clinical efficacy of SCIT treatment regimens.

1,25(OH)2VitD3 supplementation enhances suppression of grass pollen-induced allergic asthma by subcutaneous and sublingual immunotherapy in a mouse model.

Allergen specific immunotherapy (AIT) can provide long-term alleviation of symptoms for allergic disease but is hampered by suboptimal efficiency. We and others have previously shown that 1,25(OH)2-VitaminD3 (VitD3) can improve therapeutic efficacy of AIT. However, it is unknown whether VitD3 supplementation has similar effects in sublingual and subcutaneous immunotherapy. Therefore, we aimed to test VitD3 supplementation in both grass pollen (GP) subcutaneous-IT (SCIT) and sublingual-IT (SLIT) in a mouse model for allergic airway inflammation. To this end, GP-sensitized BALB/c mice received GP-SCIT or GP-SLIT with or without 10 ng VitD3, followed by intranasal GP challenges and measurement of airway hyperresponsiveness (AHR) and inflammation. VitD3 supplementation of GP-SCIT resulted in enhanced induction of GP-specific (sp)-IgG2a and suppression of spIgE after challenge. In addition, eosinophil numbers were reduced and levels of IL10 and Amphiregulin were increased in lung tissue. In GP-SLIT, VitD3 supplementation resulted in enhanced sp-IgG2a levels in serum, enhanced suppression of eosinophils and increased IL10 levels in lung tissue, as well as suppression of AHR to methacholine. These data show that VitD3 increases efficacy of both SCIT and SLIT, by enhancing induction of blocking antibodies and suppression of airway inflammation, underscoring the relevance of proficient VitD3 levels for successful AIT.

Cellular senescence impairs the reversibility of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) in congenital cardiac shunts can be reversed by hemodynamic unloading (HU) through shunt closure. However, this reversibility potential is lost beyond a certain point in time. The reason why PAH becomes irreversible is unknown. In this study, we used MCT+shunt-induced PAH in rats to identify a dichotomous reversibility response to HU, similar to the human situation. We compared vascular profiles of reversible and irreversible PAH using RNA sequencing. Cumulatively, we report that loss of reversibility is associated with a switch from a proliferative to a senescent vascular phenotype and confirmed markers of senescence in human PAH-CHD tissue. In vitro, we showed that human pulmonary endothelial cells of patients with PAH are more vulnerable to senescence than controls in response to shear stress and confirmed that the senolytic ABT263 induces apoptosis in senescent, but not in normal, endothelial cells. To support the concept that vascular cell senescence is causal to the irreversible nature of end-stage PAH, we targeted senescence using ABT263 and induced reversal of the hemodynamic and structural changes associated with severe PAH refractory to HU. The factors that drive the transition from a reversible to irreversible pulmonary vascular phenotype could also explain the irreversible nature of other PAH etiologies and provide new leads for pharmacological reversal of end-stage PAH.

Ciclosporin does not influence bone marrow-derived cell differentiation to myofibroblasts early after renal ischemia/reperfusion.

BACKGROUND: Ischemia/reperfusion injury (IRI) is a risk factor for the development of interstitial fibrosis. Previously we had shown that after renal IRI, bone marrow-derived cells (BMDC) can differentiate to interstitial myofibroblasts. Here we hypothesized that the immunosuppressant ciclosporin A (CsA), known for its profibrotic side effect, promotes myofibroblast differentiation of BMDC in the postischemic kidney. METHODS: Using a model of unilateral renal IRI in rats reconstituted with R26-human placental alkaline phosphatase transgenic bone marrow, CsA was administered in a previously defined critical window for differentiation of BMDC to myofibroblasts. We evaluated fibrotic changes in the kidney and myofibroblast differentiation of BMDC on day 14 after CsA treatment. RESULTS: CsA treatment for 14 days led to increased transforming growth factor-beta transcript levels and collagen III deposition in the postischemic kidney. However, neither the total number of alpha-smooth-muscle-actin-positive interstitial myofibroblasts, nor the bone marrow-derived fraction thereof was affected by CsA administration, irrespective of dosage and duration of treatment. CONCLUSIONS: In the critical postischemic window of BMDC differentiation to myofibroblasts, CsA did not promote BMDC differentiation to myofibroblasts, suggesting that, in the clinical setting, CsA is not involved in myofibroblastic differentiation of BMDC.

Cryoinjury: a model of myocardial regeneration.

INTRODUCTION: Although traditionally adult cardiomyocytes are thought to be unable to divide, recent observations provide evidence for cardiomyocyte proliferation after myocardial injury. Myocardial cryoinjury has been shown to be followed by neovascularization. We hypothesize that, in addition to neovascularization, cardiomyocyte proliferation after myocardial cryoinjury contributes to regeneration. METHOD: Cryolesions were applied to the left ventricle of mouse hearts. Inflammatory cell infiltration (F4/80, neutrophils), neovascularization (CD31), and cardiomyocyte proliferation (5-bromo-2-deoxyuridine, Ki-67, mitotic spindle) were determined at different time points (2-70 days) after cryoinjury. RESULTS: Between Days 7 and 14 after injury, a 150- and 280-fold increase in number of proliferating cardiomyocytes was observed, as compared to controls. At the same time, numerous proliferating capillaries were found in between the proliferating cardiomyocytes. Presence of high numbers of macrophages in the cryolesion preceded and coincided with this proliferation. The area of cryolesion decreased significantly between Days 7 (23+/-5%) and 14 (8+/-2%) after cryoinjury. Moreover, regeneration of viable, nonhypertrophied myocardium was observed. After 14 days, cardiomyocyte proliferation decreased to numbers observed in controls, and concomitantly, the number of macrophages strongly decreased. CONCLUSION: Our data show that adult cardiomyocytes proliferate in sufficiently high numbers to effectuate myocardial regeneration after left ventricular cryoinjury in mice.

Stem cell-related cardiac gene expression early after murine myocardial infarction.

OBJECTIVE: Clinical experimental stem cell therapy after myocardial infarction appears feasible, but its use has preceded the understanding of the working mechanism. The ischemic recipient cardiac environment is determinative for the attraction and subsequent fate of stem cells. Here, we studied expression levels of genes that are anticipated to be essential for adequate stem cell-based cardiac repair at various time-points during the 1 month period following myocardial infarction (MI). METHODS: Gene expression in the hearts of mice that underwent MI by permanent or transient (30 min) ligation of the coronary artery was monitored using quantitative RT-PCR analysis of mRNA isolated from whole heart sections as well as from specific, laser micro-dissected, regions of sections. Protein expression was performed by immunohistochemical stainings and Western blot analysis. RESULTS: Many inflammatory genes were highly expressed for at least 1 week after MI. The expression of pro-angiogenic genes such as bFGF, VEGF-A and VEGF-R2 changed only marginally post-MI. Markers used to test stem cell gene expression remained unchanged post-MI with the exception of G-CSF and GM-CSF, which are genes that are also known to enhance the inflammatory response. Analysis of micro-dissected regions revealed that SDF-1, SCF (both stem cell attractants) and VEGF-R2 (involved in angiogenesis) gene expression was slightly decreased especially in the infarcted region. CONCLUSION: Genes that are generally considered to participate in stem cell-related processes and angiogenesis were not upregulated after MI, whereas the inflammatory gene expression dominated. Modulation of this imbalance might be of value for stem cell-mediated therapy.

Biomaterial Encapsulation Is Enhanced in the Early Stages of the Foreign Body Reaction During Conditional Macrophage Depletion in Transgenic Macrophage Fas-Induced Apoptosis Mice.

Macrophages are pivotal cells during the foreign body reaction (FBR), as they orchestrate the proinflammatory microenvironment inside and around biomaterials by secretion of inflammatory mediators. Furthermore, they are responsible for the degradation of biomaterials and are thought to instruct the fibroblasts that generate a fibrous capsule around implanted biomaterials. In this study, we investigated the events during the FBR when macrophages are not present. Hexamethylenediisocyanate crosslinked collagen scaffolds were implanted in "Macrophage Fas-Induced Apoptosis" mice, which allow "on demand" macrophage depletion. We observed that macrophage depletion completely inhibited inflammatory ingrowth into the scaffolds and resulted in an increased capsule size. Quantitative polymerase chain reaction analysis revealed decreased expression levels of proinflammatory mediators such as TNFα and IL1ß, and increased expression levels of collagens and fibroblast-stimulating growth factors such as EGF, FGF1, FGF2, and TGFα. Our results indicate that macrophages are indeed crucial for the generation of a proinflammatory microenvironment inside implanted biomaterials, leading to inflammatory ingrowth. In contrast, macrophages do not appear to be important for the generation of a fibrous capsule around implanted biomaterials. In fact, our data suggest that the macrophages present in the capsule might instruct the surrounding fibroblasts to produce less fibroblast-stimulating factors and less collagens.

The enzymatic degradation of scaffolds and their replacement by vascularized extracellular matrix in the murine myocardium.

Replacement of injured myocardium by cell-based degradable scaffolds is a novel approach to regenerate myocardium. Understanding the foreign body reaction (FBR) induced by the scaffold is requisite to predict unwanted site effects or implant failure. We evaluated the FBR against a biodegradable scaffold applied on injured myocardium in mice. Cryolesions and collagen type I scaffolds (Col-I) were applied to the left ventricle of mice. Cell infiltration, neovascularization, collagen deposition, matrix metalloproteinase (MMP-8) expression, enzymatic activity and scaffold degradation were determined at different time points (2-70 days). Infiltration of mainly macrophages, neutrophils and blood vessels was completed within 14 days. High numbers of neutrophils accumulated around the Col-I fibers and degradation of Col-I fibers into small fragments was observed on day 14. Active MMP-8 co-localized with the neutrophils on day 14, indicating enzymatic degradation of Col-I by neutrophil collagenase. Highly vascularized extracellular matrix remained at day 70. No differences were observed in the FBR to Col-I after application on healthy or injured myocardium. The FBR had no adverse effects on the adjacent myocardial tissue. In conclusion, cardiac scaffolds are degraded by MMP-8 and replaced by vascularized extracellular matrix during the FBR on injured myocardium.

The correlation between difference in foreign body reaction between implant locations and cytokine and MMP expression.

The foreign body reaction (FBR) differs between subcutaneously and supra-epicardially implanted materials. We hypothesize that this is a result of differences in cytokine, chemokine and matrix metalloproteinase (MMP) dynamics. Therefore we applied collagen disks subcutaneously and on the epicardium in mice and analyzed the FBR from day 1 to 21. Both the influx of leukocytes and implant degradation were higher in supra-epicardially implanted collagen than in subcutaneously implanted material. This correlated with a higher gene expression of pro-inflammatory cytokines such as IL-1 and IL-6, and a lower expression of the anti-inflammatory cytokine IL-10. Furthermore, the higher supra-epicardial expression of PMN attractants CXCL1/KC and CXCL2/MIP2 correlated with a higher and prolonged PMN influx. The gene expression levels of collagen degrading MMPs, i.e. MMP8, MMP13 and MMP14 were similar in subcutaneous and supra-epicardial disks. However, the activity of these enzymes was markedly higher supra-epicardially. In addition, the MMP9 expression was higher supra-epicardially, suggesting a role for this enzyme in the degradation process. In conclusion, a strong pro-inflammatory milieu is generated after supra-epicardial implantation that enables prolonged PMN presence and activation. This, together with the high supra-epicardial MMP9 level, could explain the observed difference in Col-I degradation between locations.

Increased inflammatory response and neovascularization in reperfused vs. non-reperfused murine myocardial infarction.

INTRODUCTION: Fundamental knowledge of the inflammatory response after myocardial infarction (MI) is indispensable for intervention toward cardiac regeneration. Although reperfusion is preferred as clinical therapy, for basic research, also permanent ligation MI models are widely used. METHODS: In this report, we pathohistologically compared the kinetics of the inflammatory and angiogenic response after MI induced by permanent ligation or ligation followed by reperfusion of the left anterior descending coronary artery in mice. RESULTS: Permanent ligation resulted in a higher mortality rate accompanied by increased left ventricular dilatation and more progressive wall thinning. However, reperfused infarcts showed higher inflammatory cell influx. Neutrophil numbers were higher after reperfusion post-MI, although their presence was prolonged after ligation. Also, the number of macrophages after reperfusion was continuously higher, but the course of macrophage influx was comparable in both models. The number of lymphocytes was low in both models. Only the peak in myofibroblast numbers at 7 days was higher after ligation than after reperfusion. Moreover, cardiomyocyte remnants were cleared faster, and collagen deposition started earlier after reperfusion. In addition, reperfusion resulted in an increased angiogenic response, as was reflected in increased numbers of medium-sized and large vessels at 7 and 14 days post-MI. CONCLUSION: We show less adverse remodeling together with a higher presence of inflammatory cells and enhanced neovascularization in reperfused MI. These differences between non-reperfused and reperfused MI should be taken into consideration for experimental use of MI models.

Donor brain death aggravates chronic rejection after lung transplantation in rats.

BACKGROUND: Many recipients of lung transplants from brain-dead donors develop bronchiolitis obliterans, a manifestation of chronic rejection. It has been shown that brain death increases inflammatory mediators and accelerates acute rejection in kidney, liver, and heart transplants. In this study, the authors investigated the hypothesis that brain death increases inflammatory mediators in the donor lung and subsequently aggravates chronic rejection of the lungs after transplantation in rats. METHODS: Brain death was induced in F344 rats by inflation of a subdurally placed balloon catheter. After 6 hr, donor lungs were assessed for influx of leukocytes, expression of cell adhesion molecules, and cytokine mRNA expression. For assessment of the lung after transplantation, lungs from brain-dead F344 rats were transplanted into WKY rats. Lung function after transplantation was monitored by chest radiographs during an observation period of 100 days. At the end of this period, the lungs were histologically examined; also, cytokine mRNA expression was measured. Lungs from ventilated living donors and living donors served as controls. RESULTS: After 6 hr of brain death, influx of polymorphonuclear cells and macrophages and expression of vascular cell adhesion molecule-1 in the donor lungs was increased. After transplantation at postoperative day 100, the lung function was significantly decreased compared with allografts from living donors. In the lung allografts from brain-dead donors, histologic symptoms of chronic rejection were obvious, including severe intimal hyperplasia but without bronchiolitis obliterans. Interleukin-2 mRNA was significantly increased in allografts from brain-dead donors compared with living donors. CONCLUSIONS: This study shows that brain death induces an inflammatory response in the donor lung and subsequently aggravates chronic rejection after transplantation. This may explain the clinical difference in long-term function between lungs from cadaveric donors and living donors.

SP-A-enriched surfactant for treatment of rat lung transplants with SP-A deficiency after storage and reperfusion.

BACKGROUND: The function of pulmonary surfactant is affected by lung transplantation, contributing to impaired lung transplant function. A decreased amount of surfactant protein-A (SP-A) after reperfusion is believed to contribute to the impaired surfactant function. Surfactant treatment has been shown to improve lung transplant function, but the effect is variable. We investigated whether SP-A enrichment of surfactant improved the efficacy of surfactant treatment in lung transplantation. METHODS: Left and right lungs of Lewis rats, inflated with 50% O2, were stored for 20 hr at 8 degrees C. Surfactant in bronchoalveolar lavage fluid from right lungs was investigated after storage (n=6). Left lungs were transplanted into syngeneic recipients and treated with SP-A-deficient surfactant (n=6) or SP-A-enriched surfactant (n=6) just before reperfusion. Air was instilled into untreated lung transplants (n=6). Sham operated (n=4) and normal (n=8) animals served as controls. Lung function was measured during 1 hr of reperfusion; surfactant components in bronchoalveolar lavage fluid were measured after reperfusion. RESULTS: After storage the amount of SP-A decreased by 27%, whereas surfactant phospholipids changed minimally. After reperfusion a further decrease of SP-A was paralleled by profound changes in surfactant phospholipids. Lung transplant function, however, remained relatively good. After instillation of SP-A-enriched surfactant, PO2 values were reached that approximated sham control PO2 values, whereas after SP-A-deficient surfactant treatment, the PO2 values did not improve. CONCLUSION: Enrichment of surfactant with SP-A for treatment of lung transplants improves the efficacy of surfactant treatment.