Bernhard Sauter

Caspase-3 and Tissue Factor Expression in Lipid-Rich Plaque Macrophages Evidence for Apoptosis as Link Between Inflammation and Atherothrombosis

Background—Macrophages associated with arterial wall lipid deposition contribute to inflammatory processes. Tissue factor (TF) has been implicated in the thrombogenicity of atherosclerotic plaques. Intimal cells undergoing apoptosis have been postulated as a source for TF. However, there is only limited knowledge of cell type, plaque component, and conditions associated with TF expression and apoptosis. We examined the hypothesis that macrophages exposed to conditions of lipid-rich plaque undergo apoptosis and express TF. Methods and Results—In human carotid (n=15) and coronary (n=6) atherosclerotic plaques, TF and caspase-3 mRNA and protein expression (evaluated by in situ hybridization and immunohistochemistry) were increased significantly in lipid-rich compared with fibrous plaque components (P <0.01) and correlated with high macrophage content (P <0.05). Double-labeling studies demonstrated colocalization of TF and active caspase-3. In hyperlipidemic mice, expression of TF and active caspase-3 was observed simultaneously and colocalized in neointimal macrophages after arterial injury. In neointima of normolipidemic animals, TF and active caspase-3 were absent after arterial injury. In monocytes cultured in the presence of oxidized LDL, strong induction and colocalization of TF and active caspase-3 were found compared with baseline (P <0.05). Both antigens were significantly decreased after cotreatment with a caspase inhibitor (P <0.05) and were absent in untreated control cells. Conclusions—The expression of TF as the primary cell-associated activator of the coagulation pathway proves to be closely related to macrophages undergoing apoptosis in conditions of lipid-rich plaque, pointing to a key role of lipid content and inflammatory cell viability in determining plaque thrombogenicity.

Vascular Endothelial Growth Factor Regulates Reendothelialization and Neointima Formation in a Mouse Model of Arterial Injury

Background—The rate of reendothelialization is critical in neointima formation after arterial injury. Vascular endothelial growth factor (VEGF), a potent endothelial mitogen, has been advocated for accelerating endothelial repair and preventing intimal hyperplasia after percutaneous coronary interventions. However, the precise mechanism of action of VEGF treatment and the physiologic role of endogenous VEGF after arterial injury are not well described. To better understand the role of VEGF in arterial repair, we overexpressed both VEGF and a soluble, chimeric VEGF receptor (VEGF-trap), which binds free VEGF with high affinity, in a mouse model of arterial injury. Methods and Results—Four groups of C57BL/6 mice underwent denuding endothelial injury 1 day after systemic injection of recombinant adenovirus expressing (1) VEGF, (2) VEGF-trap, (3) VEGF plus VEGF-trap, or (4) control adenovirus. Circulating levels of adenovirus-encoded proteins were significantly elevated after gene transfer. VEGF overexpression accelerated reendothelialization and increased luminal endothelial cell proliferation 2 weeks after arterial injury (P<0.05), resulting in decreased neointima formation at 4 weeks compared with control (P<0.01). Cotreatment with VEGF-trap completely sequestered free VEGF and abrogated the beneficial effect of VEGF overexpression. Interestingly, sequestration of endogenous VEGF by VEGF-trap overexpression alone also led to delayed reendothelialization at 2 weeks (P<0.01) and increased neointima formation at 4 weeks (P<0.01). Conclusions—VEGF overexpression accelerated endothelial repair and inhibited neointima formation after arterial injury. Conversely, sequestration of exogenous and/or endogenous VEGF by VEGF-trap delayed reendothelialization and significantly increased neointima size. This demonstrates the therapeutic potential of VEGF but also emphasizes the important physiologic role of endogenous VEGF in vascular repair.

Use of the Abdominal Rectus Fascia as a Nonvascularized Allograft for Abdominal Wall Closure After Liver, Intestinal, and Multivisceral Transplantation

Introduction. Abdominal wall closure management has become an important challenge during recipient candidate selection, at the time of donor to recipient matching and during the planning of the surgical procedure for intestinal or multiorgan transplantation. Different strategies have been proposed to overcome the lack of abdominal domain: to reduce the graft size or to increase the abdominal domain. Based on the recent concept of using an acellular dermis matrix (Alloderm) and the availability of abdominal wall tissues from the same organ donor, we conceived the idea of using the fascia of the rectus muscle (FoRM) as a nonvascularized tissue allograft. Materials and Methods. This is a retrospective report of a series of 16 recipients of FoRM as part of a liver, intestinal, or multiorgan transplant procedure performed between October 2004 and May 2008 at three different transplant centers. Results. Of the 16 recipients of FoRM, all but one case was performed during their transplantation (four multivisceral, two modified multivisceral, three isolated intestine, and two livers). Five patients underwent a retransplant surgery (two livers, two multivisceral, and one isolated intestine). Abdominal wall infection was present in 7 of 16 cases. Nine patients are still alive. No deaths were related to wound infection. Long-term survival showed complete wound healing and only one ventral hernia. Discussion. The use of a nonvascularized FoRM is a novel and simple surgical option to resolve complex abdominal wall defects in liver/intestinal/multivisceral transplant recipients when it can be covered with the recipient skin.

Macrophages Transmit Potent Proangiogenic Effects of oxLDL In Vitro and In Vivo Involving HIF-1α Activation: a Novel Aspect of Angiogenesis in Atherosclerosis

Neovascularization has been linked to the progression and vulnerability of atherosclerotic lesions. Angiogenesis is increased in lipid-rich plaque. Hypoxia-inducible factor alpha (HIF-1α) is a key transcriptional regulator responding to hypoxia and activating genes, which promote angiogenesis, among them vascular endothelial growth factor (VEGF). Oxidized low-density lipoprotein (oxLDL) is generated in lipid-rich plaque by oxidative stress. It triggers an inflammatory response and was traditionally thought to inhibit endothelial cells. New data, however, suggest that oxLDL can activate HIF-1α in monocytes in a hypoxia-independent fashion. We hypothesized that HIF-1α activation in monocyte–macrophages could transmit proangiogenic effects of oxLDL linking hyperlipidemia, inflammation, and angiogenesis in atherosclerosis. First, we examined the effect of oxLDL on HIF-1α and VEGF expression in monocyte–macrophages and on their proangiogenic effect on endothelial cells in vitro in a monocyte–macrophage/endothelial co-culture model. OxLDL strongly induced HIF-1α and VEGF in monocyte–macrophages and significantly increased tube formation in co-cultured endothelial cells. HIF-1α inhibition reversed this effect. Second, we demonstrated a direct proangiogenic effect of oxLDL in an in vivo angiogenesis assay. Again, HIF-1α inhibition abrogated the proangiogenic effect of oxLDL. Third, in a rabbit atherosclerosis model, we studied the effect of dietary lipid lowering on arterial HIF-1α and VEGF expression. The administration of low-lipid diet significantly reduced the expression of both HIF-1α and VEGF, resulting in decreased plaque neovascularization. Our data point to oxLDL as a proangiogenic agent linking hyperlipidemia, inflammation, and angiogenesis in atherosclerosis. This effect is dependent on macrophages and, at least in part, on the induction of the HIF-1α pathway.

Decreased Reendothelialization and Increased Neointima Formation With Endostatin Overexpression in a Mouse Model of Arterial Injury

Background—Impaired endothelial regeneration contributes to arterial lesion formation. Endostatin is a specific inhibitor of endothelial cell growth and induces endothelial cell apoptosis. We examined the effect of endostatin overexpression on reendothelialization and neointima formation in a mouse model of arterial injury. Methods and Results—Mice underwent femoral arterial denudation and received recombinant adenovirus, expressing either murine endostatin (n=19) or control adenoviral vector (n=12), by jugular vein injection. Endostatin gene transfer resulted in high serum levels of endostatin. Strong adenoviral gene expression of &bgr;-galactosidase–expressing control vector was detected in liver tissue and was absent in the injured arterial wall at 1 week. Deposits of endostatin protein were detected along the denuded arterial wall and were not seen in the noninjured contralateral artery at 1 week. Endostatin deposits were also absent in the injured artery of control vector–treated animals. Overexpression of endostatin led to decreased reendothelialization and increased apoptosis of luminal endothelial cells 2 and 4 weeks after arterial injury (P <0.05). In addition, endostatin overexpression resulted in increased neointima formation (P <0.05). Endothelial apoptosis and neointima area correlated positively with endostatin serum levels, whereas the degree of reendothelialization correlated negatively with endostatin serum levels (P <0.05). Furthermore, poor reendothelialization correlated with increased neointima formation (P <0.05). Conclusions—In summary, decreased reendothelialization and enhanced endothelial apoptosis, in response to endostatin overexpression, were associated with increased neointima formation. These findings demonstrate that high serum levels of endostatin are capable of inhibiting endothelial regeneration and promoting arterial lesion growth in conditions of endothelial injury.

Regression of Hepatic Fibrosis After Intestinal Transplantation in Total Parenteral Nutrition Liver Disease

BACKGROUND & AIMSnHepatic fibrosis may occur in patients with intestinal failure requiring total parenteral nutrition, leading to liver dysfunction necessitating combined intestinal and liver transplantation. The decision to perform combined transplantation as opposed to an isolated intestinal transplant is based on the presence of hyperbilirubinemia, portal hypertension, and advanced hepatic fibrosis.nnnMETHODSnWe identified 4 patients who underwent isolated intestinal transplantation having significant liver fibrosis. A novel image analysis technique was applied to serial liver biopsies to more precisely quantitate posttransplantation fibrosis regression separately within both portal and centrilobular areas.nnnRESULTSnAll patients were found to have significant portal and centrilobular fibrosis regression, which occurred more rapidly in the former. Two patients had improvement in fibrosis despite infections and continuation of total parenteral nutrition, suggesting that hepatic fibrosis associated with intestinal failure may in part be related to adequate anatomic and functional bowel length.nnnCONCLUSIONSnSignificant hepatic fibrosis and liver dysfunction may regress after intestinal transplantation and fibrosis regresses more rapidly in portal areas. This suggests that some patients with intestinal failure and associated liver disease may safely undergo isolated intestinal transplant without the need for concurrent liver transplantation and its attendant higher morbidity and mortality.

Efficient infection of tumor endothelial cells by a capsid-modified adenovirus

Targeted antiangiogenic gene therapy is an attractive approach to treat metastatic cancer. However, the relative paucity of the receptors of the commonly used adenovirus serotype 5 in endothelial cells as compared with liver cells undermines the use of this vector for targeting the endothelial cells in tumors. To overcome this problem, we analyzed the ability of a hybrid Ad5/35 virus, where the serotype 5 fiber has been replaced with the fiber from serotype 35, to target tumor vasculature. Infection of human umbilical vein endothelial cells (HUVECs) with Ad5/35 at MOI 120 infected 100% of cells. In contrast, infection with Ad5 at the same MOI infected only 10% HUVECs. Ad5/35 was even more effective in transducing human aortic endothelial cells (HAECs), as infection with Ad5/35 at MOI 3.6 was sufficient to transduce 95% of cells. Gene expression analyses demonstrated that infection of HUVECs and HAECs with Ad5/35 resulted in between 1 and 3 orders of magnitude higher gene expression than infection with Ad5. Furthermore, various liver-derived cells were less infectable with Ad5/35 than Ad5, indicating a favorable toxicity profile for this virus. In a rat colon carcinoma tumor model, Ad5 was located mainly in the liver parenchyma after hepatic artery administration. In contrast, Ad5/35 was found only in the angiogenesis-rich border region of the tumor. Double immunostaining revealed that Ad5/35 colocalized with CD31 and Flk-1 positive endothelial cells. These results indicate that Ad5/35 may be useful in anticancer strategies targeting tumor endothelial cells.

Efficacy and safety of certolizumab pegol induction therapy in an unselected Crohn's disease population: Results of the FACTS survey†

Background: Switzerland was the first country to approve certolizumab pegol (Cimzia, CZP) for the treatment of patients with moderate to severe Crohns disease (CD) in September 2007. This phase IV study aimed to evaluate the efficacy and safety of CZP in a Swiss multicenter cohort of practice‐based patients. Methods: Baseline and Week 6 evaluation questionnaires were sent to all Swiss gastroenterologists in hospitals and private practices. Disease activity was assessed with the Harvey–Bradshaw Index (HBI) and adverse events were evaluated according to WHO guidelines. Results: Fifty patients (31 women, 19 men) were included; 56% had complicated disease (stricture or fistula) and 52% had undergone prior CD‐related surgery. All patients had prior exposure to systemic steroids, 96% to immunomodulators, 78% to infliximab, and 50% to adalimumab. A significant decrease in HBI was observed at Week 6 (versus Week 0) following induction therapy with CZP 400 mg subcutaneously at Weeks 0, 2, and 4 (12.6 ± 4.7 Week 0 versus 6.2 ± 4.4 Week 6, P < 0.001). Response and remission rates at Week 6 were 54% and 40%, respectively. We identified 8/11 CD patients undergoing a 50% fistula response (P = 0.021). The frequency of adverse drug reactions attributed to CZP was 6%. CZP was continued in 80% of patients beyond Week 6. Conclusions: In a population of CD patients with complicated disease behavior, CZP induced a response and remission in 54% and 40% of patients, respectively. This series provides the first evidence of the effectiveness of CZP in perianal fistulizing CD. Inflamm Bowel Dis 2010

Pretransplant immunomodulation of highly sensitized small bowel transplant candidates with intravenous immune globulin.

Presence of preformed lymphocytotoxic antibodies may represent a barrier to isolated intestinal transplantation (IITx). We developed an intravenous immunoglobulins (IVIg) based desensitization protocol for candidates with high panel-reactive antibodies (PRA). Six patients with a mean PRA of 72±22% were included in a four-level (L) protocol with escalating doses of IVIg (L1, L2), addition of mycophenolate mofetil (MMF) or plasmapheresis (L3); and anti-CD20 (Rituximab) (L4). Four of six candidates improved their PRAs (from a mean of 66.2% to 25.5%; P=0.01) and were successfully transplanted. At a mean follow-up of 8 months, number and severity of rejection episodes of protocol patients did not differ from patients with low PRA transplanted during the same period. These data support the use of IVIg to desensitize patients waiting for IITx. It increases the applicability of IITx, and reduces the waiting time and mortality on the waiting list with outcomes comparable to nonsensitized recipients.

Efficacy and safety of certolizumab pegol in an unselected crohn's disease population: 26-week data of the FACTS II survey†

Background: Certolizumab pegol (Cimzia, CZP) was approved for the treatment of Crohns disease (CD) patients in 2007 in Switzerland as the first country worldwide. This prospective phase IV study aimed to evaluate the efficacy and safety of CZP over 26 weeks in a multicenter cohort of practice‐based patients. Methods: Evaluation questionnaires at baseline, week 6, and week 26 were completed by gastroenterologists in hospitals and private practices. Adverse events were evaluated according to World Health Organization (WHO) guidelines. Results: Sixty patients (38F/22M) were included; 53% had complicated disease (stricturing or penetrating), 45% had undergone prior CD‐related surgery. All patients had prior exposure to systemic steroids, 96% to immunomodulators, 73% to infliximab, and 43% to adalimumab. A significant decrease of the Harvey‐Bradshaw Index (HBI) was observed under CZP therapy (12.2 ± 4.9 at week 0 versus 6.3 ± 4.7 at week 6 and 6.7 ± 5.3 at week 26, both P < 0.001). Response and remission rates were 70% and 40% (week 6) and 67% and 36%, respectively (week 26). The complete perianal fistula closure rate was 36% at week 6 and 55% at week 26. The frequency of adverse drug reactions attributed to CZP was 5%. CZP was continued in 88% of patients beyond week 6 and in 67% beyond week 26. Conclusions: In a population of CD patients with predominantly complicated disease behavior, CZP proved to be effective in induction and maintenance of response and remission. This series provides the first evidence of CZPs effectiveness in perianal fistulizing CD in clinical practice. (Inflamm Bowel Dis 2011;)