Robert A. Matthijsen

Inhibition of complement factor C5 protects against renal ischemia-reperfusion injury : Inhibition of late apoptosis and inflammation

Background. Complement has been implicated in the pathophysiology of renal ischemia-reperfusion (I/R) injury. However, the mechanism underlying complement-mediated renal I/R injury is thus far unknown. To investigate the involvement of complement in I/R injury, we studied the activation and deposition of complement in a murine model of renal I/R injury. Furthermore, we examined the effect of inhibition of complement-factor C5 on renal I/R injury. Methods. Mice were subjected to 45 min of unilateral ischemia and subsequent contralateral nephrectomy and reperfusion for 2, 12, or 24 hr. Mice were control treated or treated with BB5.1, a monoclonal antibody that prevents cleavage of complement factor C5, thereby preventing C5a generation and formation of the membrane attack complex (MAC). Results. Renal I/R induced extensive deposition of C3 early after reperfusion, whereas C6 and C9 deposition (MAC formation) occurred relatively late. I/R-induced complement deposition was mainly localized to tubular epithelium. Treatment with BB5.1 totally prevented MAC formation but also reduced C3 deposition. Inhibition of C5 strongly inhibited late inflammation, as measured by neutrophil influx and induction of the murine CXC chemokines macrophage inflammatory protein-2, KC, and lipopolysaccharide-induced CXC chemokine. Anti-C5 treatment furthermore abrogated late I/R-induced apoptosis, whereas early apoptosis was not affected. Moreover, BB5.1 treatment significantly protected against I/R-induced renal dysfunction. Conclusions. Renal I/R is followed by activation of the complement system and intrarenal deposition of C3 and MAC. Complement activation plays a crucial role in the regulation of inflammation and late apoptosis. Complement inhibition, by preventing C5 activation, abrogates late apoptosis and inflammation, being strongly protective against renal function loss.

Human Intestinal Ischemia-Reperfusion–Induced Inflammation Characterized : Experiences from a New Translational Model

Human intestinal ischemia-reperfusion (IR) is a frequent phenomenon carrying high morbidity and mortality. Although intestinal IR-induced inflammation has been studied extensively in animal models, human intestinal IR induced inflammatory responses remain to be characterized. Using a newly developed human intestinal IR model, we show that human small intestinal ischemia results in massive leakage of intracellular components from ischemically damaged cells, as indicated by increased arteriovenous concentration differences of intestinal fatty acid binding protein and soluble cytokeratin 18. IR-induced intestinal barrier integrity loss resulted in free exposure of the gut basal membrane (collagen IV staining) to intraluminal contents, which was accompanied by increased arteriovenous concentration differences of endotoxin. Western blot for complement activation product C3c and immunohistochemistry for activated C3 revealed complement activation after IR. In addition, intestinal IR resulted in enhanced tissue mRNA expression of IL-6, IL-8, and TNF-alpha, which was accompanied by IL-6 and IL-8 release into the circulation. Expression of intercellular adhesion molecule-1 was markedly increased during reperfusion, facilitating influx of neutrophils into IR-damaged villus tips. In conclusion, this study for the first time shows the sequelae of human intestinal IR-induced inflammation, which is characterized by complement activation, production and release of cytokines into the circulation, endothelial activation, and neutrophil influx into IR-damaged tissue.

Rapid Reversal of Human Intestinal Ischemia-Reperfusion Induced Damage by Shedding of Injured Enterocytes and Reepithelialisation

Background Intestinal ischemia-reperfusion (IR) is a phenomenon related to physiological conditions (e.g. exercise, stress) and to pathophysiological events (e.g. acute mesenteric ischemia, aortic surgery). Although intestinal IR has been studied extensively in animals, results remain inconclusive and data on human intestinal IR are scarce. Therefore, an experimental harmless model for human intestinal IR was developed, enabling us to clarify the sequelae of human intestinal IR for the first time. Methods and Findings In 30 patients undergoing pancreatico-duodenectomy we took advantage of the fact that in this procedure a variable length of jejunum is removed. Isolated jejunum (5 cm) was subjected to 30 minutes ischemia followed by reperfusion. Intestinal Fatty Acid Binding Protein (I-FABP) arteriovenous concentration differences across the bowel segment were measured before and after ischemia to assess epithelial cell damage. Tissue sections were collected after ischemia and at 25, 60 and 120 minutes reperfusion and stained with H&E, and for I-FABP and the apoptosis marker M30. Bonferronis test was used to compare I-FABP differences. Mean (SEM) arteriovenous concentration gradients of I-FABP across the jejunum revealed rapidly developing epithelial cell damage. I-FABP release significantly increased from 290 (46) pg/ml before ischemia towards 3,997 (554) pg/ml immediately after ischemia (p<0.001) and declined gradually to 1,143 (237) pg/ml within 1 hour reperfusion (p<0.001). Directly after ischemia the intestinal epithelial lining was microscopically normal, while subepithelial spaces appeared at the villus tip. However, after 25 minutes reperfusion, enterocyte M30 immunostaining was observed at the villus tip accompanied by shedding of mature enterocytes into the lumen and loss of I-FABP staining. Interestingly, within 60 minutes reperfusion the epithelial barrier resealed, while debris of apoptotic, shedded epithelial cells was observed in the lumen. At the same time, M30 immunoreactivity was absent in intact epithelial lining. Conclusions This is the first human study to clarify intestinal IR induced cell damage and repair and its direct consequences. It reveals a unique, endogenous clearing mechanism for injured enterocytes: rapid detachment of damaged apoptotic enterocytes into the lumen. This process is followed by repair of the epithelial continuity within an hour, resulting in a normal epithelial lining.

Enterocyte Shedding and Epithelial Lining Repair Following Ischemia of the Human Small Intestine Attenuate Inflammation

Background Recently, we observed that small-intestinal ischemia and reperfusion was found to entail a rapid loss of apoptotic and necrotic cells. This study was conducted to investigate whether the observed shedding of ischemically damaged epithelial cells affects IR induced inflammation in the human small gut. Methods and Findings Using a newly developed IR model of the human small intestine, the inflammatory response was studied on cellular, protein and mRNA level. Thirty patients were consecutively included. Part of the jejunum was subjected to 30 minutes of ischemia and variable reperfusion periods (mean reperfusion time 120 (±11) minutes). Ethical approval and informed consent were obtained. Increased plasma intestinal fatty acid binding protein (I-FABP) levels indicated loss in epithelial cell integrity in response to ischemia and reperfusion (p<0.001 vs healthy). HIF-1α gene expression doubled (p = 0.02) and C3 gene expression increased 4-fold (p = 0.01) over the course of IR. Gut barrier failure, assessed as LPS concentration in small bowel venous effluent blood, was not observed (p = 0.18). Additionally, mRNA expression of HO-1, IL-6, IL-8 did not alter. No increased expression of endothelial adhesion molecules, TNFα release, increased numbers of inflammatory cells (p = 0.71) or complement activation, assessed as activated C3 (p = 0.14), were detected in the reperfused tissue. Conclusions In the human small intestine, thirty minutes of ischemia followed by up to 4 hours of reperfusion, does not seem to lead to an explicit inflammatory response. This may be explained by a unique mechanism of shedding of damaged enterocytes, reported for the first time by our group.

Macrophage-Specific Expression of Mannose-Binding Lectin Controls Atherosclerosis in Low-Density Lipoprotein Receptor–Deficient Mice

Background— With consideration of the central role of the innate immune system in atherogenesis and mannose-binding lectin (MBL) as an innate regulator of immunity, the role of MBL in experimental and human atherosclerosis was assessed. Methods and Results— With the use of immunohistochemistry and polymerase chain reaction, deposition and gene expression of MBL-A and -C were assessed in murine atherosclerosis from mice deficient for the low-density lipoprotein receptor (LDLR−/−) after 10 or 18 weeks of high-fat feeding. MBL was present and was produced in 10-week-old lesions, whereas deposition and gene expression were minimal after 18 weeks of high-fat feeding and absent in healthy vasculature. Interestingly, deposition of MBL-A and -C differed: MBL-A predominantly localized in upper medial layers, whereas MBL-C was found in and around intimal macrophages. To further study the role of local MBL production by monocytic cells in atherosclerosis, LDLR−/− mice with MBL-A and -C−/− monocytic cells were construed by bone marrow transplantation. Mice carrying MBL-A and -C double deficient macrophages had increased (30%) atherosclerotic lesions compared with wild-type controls (P=0.015) after 10 weeks of high-fat diet. Subsequently, analysis of MBL deposition and gene expression in advanced human atherosclerotic lesions revealed the presence of MBL protein in ruptured but not stable atherosclerotic lesions. Putatively in agreement with murine data, no MBL gene expression could be detected in advanced human atherosclerotic lesions. Conclusions— These results are the first to show that MBL is abundantly present and locally produced during early atherogenesis. Local MBL expression, by myeloid cells, is shown to critically control development of atherosclerotic lesions.

Lysophosphatidic Acid Prevents Renal Ischemia-Reperfusion Injury by Inhibition of Apoptosis and Complement Activation

Renal ischemia-reperfusion (I/R) injury is an important cause of acute renal failure as observed after renal transplantation, major surgery, trauma, and septic as well as hemorrhagic shock. We previously showed that the inhibition of apoptosis is protective against renal I/R injury, indicating that apoptotic cell-death is an important feature of I/R injury. Lysophosphatidic acid (LPA) is an endogenous phospholipid growth factor with anti-apoptotic properties. This tempted us to investigate the effects of exogenous LPA in a murine model of renal I/R injury. LPA administered at the time of reperfusion dose dependently inhibited renal apoptosis as evaluated by the presence of internucleosomal DNA cleavage. I/R-induced renal apoptosis was only present in tubular epithelial cells with evident disruption of brush border as assessed by immunohistochemistry for active caspase-7 and filamentous actin, respectively. LPA treatment specifically prevented tubular epithelial cell apoptosis but also reduced the I/R-induced loss of brush-border integrity. Besides, LPA showed strong anti-inflammatory effects, inhibiting the renal expression of tumor necrosis factor-alpha and abrogating the influx of neutrophils. Next, LPA dose dependently inhibited activation of the complement system. Moreover, treatment with LPA abrogated the loss of renal function in the course of renal I/R. This study is the first to show that administration of the phospholipid LPA prevents I/R injury, abrogating apoptosis and inflammation. Moreover, exogenous LPA is capable of preventing organ failure because of an ischemic insult and thus may provide new means to treat clinical conditions associated with I/R injury in the kidney and potentially also in other organs.

C1-Esterase Inhibitor Protects Against Neointima Formation After Arterial Injury in Atherosclerosis-Prone Mice

Background— Although activation of the complement system has been implicated in the progression of human atherosclerosis, its function during arterial remodeling after injury has not been investigated. Here, we examined the contribution of the complement cascade to neointima formation in apolipoprotein E–deficient mice using a C1-esterase inhibitor (C1-inhibitor). Methods and Results— Apolipoprotein E–deficient mice fed an atherogenic diet were subjected to wire-induced endothelial denudation of the carotid artery and treated with C1-inhibitor (Berinert; 15 IU IV) or vehicle perioperatively and subsequently every 2 days. The effectiveness of C1-inhibitor treatment was confirmed by measurement of plasma C1-inhibitor activity. A significant reduction in serum triglyceride levels was observed in C1-inhibitor–treated mice, whereas cholesterol levels did not differ. After 3 weeks, neointimal area was significantly reduced in C1-inhibitor–treated mice versus controls, whereas medial area was unaltered. This was associated with a significant decrease in neointimal and medial macrophage and CD3+ T-cell content. Expression of C3 mRNA was significantly reduced in plaques of C1-inhibitor–treated mice 10 days after injury, as assessed by reverse-transcription polymerase chain reaction. The peak in serum C3 levels after injury was markedly downregulated by C1-inhibitor, as evidenced by ELISA. Immunohistochemistry revealed strong expression of C3 and C3c, which colocalized to plaque macrophages and was reduced in C1-inhibitor–treated mice. C1-inhibitor impaired monocyte arrest on activated endothelium and platelets under flow conditions in vitro and leukocyte recruitment to carotid arteries 1 day after injury in vivo. Conclusions— C1-inhibitor limits neointimal plaque formation and inflammation. This may involve blockade of complement activation, inhibition of leukocyte recruitment, and reduced triglyceride levels, thus providing a multimodal approach to treat arterial disease.

A New Model to Study Intestinal Ischemia-Reperfusion Damage in Man

BACKGROUND This report describes a human in vivo ischemia reperfusion (IR) model of the small intestine. Animal models of intestinal IR are indispensable for our understanding of sequelae of IR induced organ damage. However, a functional experimental IR model of the human small intestine, allowing for translational research, can be considered critical for our pathophysiologic understanding of intestinal IR in man. MATERIALS AND METHODS Patients with a healthy gut undergoing abdominal surgery with a Roux-Y or similar reconstruction were included, creating the opportunity to study IR of an isolated jejunal segment in a harmless model. RESULTS Ischemia was induced by nontraumatic vascular clamping followed by reperfusion. This model can be adapted using variable ischemia and reperfusion times. Similarly, tissue and plasma can be collected at any given time point during ischemia until end of reperfusion, only determined by progress of the original, intended surgical procedure. CONCLUSION A unique and harmless human IR model of the jejunum was created, which enables the study of acute damage to the epithelial lining and its subsequent repair mechanisms.

Autologous transplantation of ischemically injured kidneys in pigs.

BACKGROUND Expansion of the organ donor pool can be obtained through novel interventions attenuating ischemic acute kidney injury, which will enable the use of kidneys that suffered prolonged ischemia. In basic science, new therapeutic targets are identified that should be tested in a relevant large animal model before use in human kidney transplantation. MATERIALS AND METHODS The current paper provides a detailed description of the technique of autologous transplantation of ischemically injured kidneys in pigs with special emphasis on perioperative care. RESULTS The animal model was validated by showing that renal function after transplantation was proportional to the duration of warm ischemia before organ recovery. The extent of renal dysfunction was reproducible following kidney transplantations with the same warm ischemia time. CONCLUSIONS Our experience may reduce the learning curves of other research groups taking an interest in the model and improve preclinical testing of novel interventions that modulate renal ischemia and reperfusion injury in kidney transplantation.