John E. Rectenwald

Direct Evidence for Cytokine Involvement in Neointimal Hyperplasia

BackgroundTumor necrosis factor-&agr; (TNF-&agr;) and interleukin 1 (IL-1) are proximal inflammatory cytokines that stimulate expression of adhesion molecules and induce synthesis of other proinflammatory cytokines. In addition, TNF-&agr; and IL-1 influence vascular smooth muscle cell migration and proliferation in vitro. In view of the inflammatory nature of neointimal hyperplasia (NIH), we tested the hypothesis that endogenous TNF-&agr; and IL-1 modulate low shear stress–induced NIH. Methods and ResultsMice underwent unilateral common carotid artery (CCA) ligation. Low shear stress in the patent ligated CCA has previously been shown to result in remodeling and NIH. Reverse transcriptase–polymerase chain reaction for TNF-&agr; and IL-1&agr; mRNA demonstrated both TNF-&agr; and IL-1&agr; mRNA in ligated CCAs, whereas normal and sham-operated CCAs had none. Mice lacking functional TNF-&agr; (TNF−/−) developed 14-fold less neointimal area than WT controls (P <0.05). p80 IL-1 type I receptor knockout (IL-1RI−/−) mice tended to develop less (7-fold, P >0.05) neointimal area than WT controls. Furthermore, no IL-1&agr; mRNA expression was detected in CCAs from TNF−/− mice; however, TNF-&agr; mRNA expression was found in the IL-1RI−/− mice. Mice that overexpress membrane-bound TNF-&agr; but produce no soluble TNF-&agr; display an accentuated fibroproliferative response to low shear stress (P <0.05). ConclusionsThese results directly demonstrate that TNF-&agr; and IL-1 modulate NIH induced by low shear stress. NIH can proceed by way of soluble TNF-&agr;–independent mechanisms. Specific anti–TNF-&agr; and anti–IL-1 therapies may lessen NIH.

Arteriogenesis Proceeds via ICAM-1/Mac-1- Mediated Mechanisms

Monocyte adhesion to shear stress–activated endothelium stands as an important initial step during arteriogenesis (collateral artery growth). Using multiple approaches, we tested the hypothesis that monocyte adhesion via intercellular adhesion molecule-1 (ICAM-1) and selectin interactions is essential for adaptive arteriogenesis. Forty-eight New Zealand White rabbits received either solvent, monocyte chemoattractant protein-1 (MCP-1) alone, MCP-1 plus ICAM-mab, or MCP-1 plus an IgG2a isotype control via osmotic minipumps. After 7 days, collateral conductance was evaluated: solvent 4.01 (mL/min per 100 mm Hg), MCP-1 plus ICAM-mab 8.04 (versus solvent P =NS), and MCP-1 alone 33.11 (versus solvent P <0.05). Furthermore, the right femoral arteries of ICAM-1−/−, Mac-1−/− and mice having defective selectin interactions (FT4/7−/−) as well as their corresponding controls were ligated. One week later, perfusion ratios were determined by the use of fluorescent microspheres. FT4/7−/− mice did not show any significant difference in perfusion restoration whereas ICAM-1−/− and Mac-1−/− mice had a significant reduction in arteriogenesis as compared with matching controls (FT4/7-WT 37±9%, FT4/7−/− 32±3%, P =0.31; C57BL/6J 59±9%, ICAM-1−/− 36±8%, P <0.05; Mac-1−/− 42±3%, P <0.05). ICAM-1/Mac-1–mediated monocyte adhesion to the endothelium of collateral arteries is an essential step for arteriogenesis, whereas this process can proceed via selectin interaction independent mechanisms. Furthermore, in vivo treatment with monoclonal antibodies against ICAM-1 totally abolishes the stimulatory effect of MCP-1 on collateral artery growth, suggesting that the mechanism of the MCP-1–induced arteriogenesis proceeds via the localization of monocytes rather than the action of the MCP-1 molecule itself.

Direct Evidence for Tumor Necrosis Factor-α Signaling in Arteriogenesis

Background—Arteriogenesis serves as an efficient mechanism for flow restoration after arterial occlusion. This process is associated with inflammatory mediators such as tumor necrosis factor-&agr; (TNF-&agr;), although their role in arteriogenesis remains unclear. We hypothesized that arteriogenesis is reduced in mice lacking functional TNF-&agr; or p55 receptor. To test this hypothesis, we developed a novel microsphere-based murine model of hindlimb perfusion measurement. Methods and Results—Unilateral femoral arteries of nude (n=9), TNF-&agr;−/− (n=9), TNF-&agr; receptor p55−/− (n=8), and p75−/− (n=8) mice as well as their appropriate genetic background controls were occluded. The nude mice underwent laser Doppler hindlimb flux measurements preoperatively, postoperatively, and after 7 days. Seven days after ligation, all animals underwent tissue perfusion determinations using fluorescent microspheres. Laser Doppler findings confirmed acute decrease in flux with falsely normal values after 1 week. Microsphere results from control mice showed perfusion restoration to values ≈50% of normal within 7 days. TNF-&agr;−/− mice demonstrated a significant reduction (45.1%) in collateral artery perfusion compared with controls (TNF-&agr;−/− 22.4±5.1% versus B6x129 49.7±9.3%;P <0.01). p55−/− mice exhibited an almost identical 45.8% reduction in collateral artery formation (p55−/− 28.3±4.3% versus C57BL/6J 61.8±9.1%;P <0.01), whereas p75−/− mice were equivalent to controls (p75−/− 54.5±5.5%;P =0.13). Conclusions—Microsphere techniques in mice offer a tool for the molecular dissection of arteriogenesis mechanisms. These results suggest that TNF-&agr; positively modulates arteriogenesis probably via signaling through its p55 receptor.

TNF-α Receptor Signaling and IL-10 Gene Therapy Regulate the Innate and Humoral Immune Responses to Recombinant Adenovirus in the Lung

Recombinant adenovirus-mediated gene therapy has demonstrated great promise for the delivery of genes to the pulmonary epithelium. However, dose-dependent inflammation and local immune responses abbreviate transgene expression. The purpose of these studies was to determine the role of TNF-α and individual TNF receptor signaling to adenovirus clearance and immune responses, and whether coexpression of human IL-10 could reduce inflammation and extend the duration of transgene expression in the lung. β-Galactosidase expression in mice receiving intratracheal instillation of Adv/β-gal (adenovirus construct expressing β-galactosidase) was transient (less than 14 days), but a significant early increase of β-galactosidase expression was seen in mice lacking either or both TNF-α receptors. Absence of TNF-α or the p55 receptor significantly attenuated the Ab response to both adenovirus and β-galactosidase. Human IL-10 expression in the lung suppressed local TNF-α production following AdV/hIL-10 (adenovirus construct expressing human IL-10) delivery, but did not lead to increased or prolonged transgene expression when coexpressed with β-galactosidase. Expression of human IL-10 following AdV/hIL-10 instillation extended at least 14 days, was nonimmunogenic, and suppressed the development of neutralizing Abs against adenoviral proteins as well as against human IL-10. We conclude that TNF-α signaling through both the p55 and p75 receptor plays important roles in the clearance of adenoviral vectors and the magnitude of the humoral immune response. Additionally, although coexpression of human IL-10 with β-galactosidase had only modest effects on transgene expression, we demonstrate that AdV/hIL-10 is well tolerated, has extended expression compared with β-galactosidase, and is nonimmunogenic in the lung.

Bioglass attenuates a proinflammatory response in mouse peritoneal endotoxicosis.

Bioglass® is a bioactive, resorbable ceramic particle that was developed to assure binding to living tissues. Bioglass® is currently employed to fill osseus defects in oral surgery, and it possesses both unique anti-inflammatory and antimicrobial properties. In an effort to determine whether Bioglass® may be useful as an adjunct anti-inflammatory device in local inflammatory processes, we examined whether exposure of the peritoneal cavity to Bioglass® would induce a pro- or anti-inflammatory response, and then modulate a subsequent proinflammatory response to endotoxin. Three- to fifty-milligram doses of 5 &mgr;m Bioglass® were administered intraperitoneally in C57BL/6 mice. Total leukocyte, myeloperoxidase, and cytokine levels in the peritoneal wash fluid were determined. In addition, the peritoneal cavity was pre-exposed to Bioglass®, and was then subjected to a subsequent endotoxin administration. All doses of Bioglass® were found to induce a significant peritoneal IL-6 response; however, Bioglass® did not induce a TNF-&agr;, IL-1&agr;, IL-10, or a white cell recruitment into the peritoneal lavage fluid. Pretreatment of the peritoneal cavity with Bioglass® produced a transient reduction in the proinflammatory response to endotoxin. We conclude that exposure to Bioglass® produces an IL-6 response without concurrent expression of TNF-&agr; or IL-1&agr;. Bioglass® appears to transiently suppress the inflammatory response to endotoxin, possibly through the early induction of IL-6. These findings suggest that Bioglass® may offer a unique approach in modifying the inflammatory response in local tissue compartments.

External-Beam Radiation Therapy for Improved Dialysis Access Patency: Feasibility and Early Safety

Abstract Rectenwald, J. E., Pretus, H. A., Seeger, J. M., Huber, T. S., Mendenhall, N. P., Zlotecki, R. A., Palta, J. R., Li, Z. F., Hook, S. Y., Sarac, T. P., Welborn, M. B., Klingman, N. V., Abouhamze, Z. S. and Ozaki, C. K. External-Beam Radiation Therapy for Improved Dialysis Access Patency: Feasibility and Early Safety. Radiat. Res. 156, 53–60 (2001). Prosthetic dialysis access grafts fail secondary to neointimal hyperplasia at the venous anastomosis. We hypothesized that postoperative single-fraction external-beam radiation therapy to the venous anastomosis of hemodialysis grafts can be used safely in an effort to improve access patency. Dogs (n = 8) underwent placement of expanded polytetrafluoroethylene grafts from the right carotid artery to the left jugular vein. Five dogs received single-fraction external-beam photon irradiation (8 Gy) to the venous anastomosis after surgery. Controls were not irradiated. Shunt angiograms were completed 3 and 6 months postoperatively. Anastomoses, mid-graft, and the surrounding tissues were analyzed. Immunohistochemistry for smooth muscle cell α-actin, proliferating cellular nuclear antigen (PCNA), and apoptosis was performed. Incisions healed well, though all animals developed wound seromas. One control suffered graft thrombosis 4 months postoperatively. Angiography/histology confirmed severe neointimal hyperplasia at the venous anastomosis. The remaining seven dogs developed similar amounts of neointimal hyperplasia. PCNA studies showed no accelerated fibroproliferative response at irradiated anastomoses compared to controls. Skin incisions and soft tissues over irradiated anastomoses revealed no radiation-induced changes or increase in apoptosis. Thus we conclude that postoperative single-fraction external-beam irradiation of the venous anastomosis of a prosthetic arteriovenous graft that mimics the situation in humans is feasible and safe with regard to early wound healing.

Emerging Evidence of a More Complex Role for Proinflammatory and Antiinflammatory Cytokines in the Sepsis Response

Since the original discovery and cloning of tumor necrosis factor-α (TNFα) and interleukin-1 (IL-1), our understanding of the underlying role that these and other cytokines play in the sepsis response has greatly evolved.The original concept that the sepsis response is a result of a linear cytokine cascade induced by TNFα and IL-1 has given way to the appreciation that sepsis syndromes often result from a more complex interplay between proinflammatory cytokines, antiinflammatory cytokines, and cytokine antagonists. The proinflammatory cytokine-dominated, systemic inflammatory response syndrome is likely an episodic or transient occurrence; and many septic patients present with a compensatory antiinflammatory cytokine response dominated by the release of antiinflammatory cytokines and cytokine antagonists, leading to immune suppression. There is also growing appreciation that other members of the TNFα superfamily, including Fas ligand (FasL) and glucocorticoids, play an increasingly important role in the loss of immune cells during sepsis through apoptotic processes. The cytokine component of the innate immune response to sepsis plays a complex role not only in the inflammatory response syndrome but also in determining the nature and magnitude of the acquired immune response.