The modern era of cardiac surgery is largely considered to have begun in the animal research laboratories. Today, animal models continue to be used for the study of cardiovascular diseases and are required for the preclinical assessment of pharmaceuticals, mechanical devices, therapeutic procedures, and/or continuation therapies. This chapter was designed to provide readers and potential investigators with important background information necessary for the process of matching an experimental hypothesis to an animal species that will serve as an appropriate model for studying a specific cardiovascular disease or for testing a given medical device. A review of the current animal models used in cardiac research is provided and arranged by disease state. Critical factors to consider when choosing an appropriate animal model including cost, reproducibility, and degree of similarity of the model to human disease are discussed. Thus, this chapter can be utilized as a practical guide for planning of research protocols.

Objective: Novel mitral valve devices must pass rigorous preclinical testing to be approved for clinical use. This study aims to validate an appropriate preclinical control model of mitral valve replacement as a foundation for testing of novel valves, using mitral valves currently in clinical use: the Carpentier-Edwards Perimount (CEP) Magna (7300TFX) and Plus (6900P) valves. Methods: Via 4th space thoracotomy, 15 sheep underwent implantation of either a CEP Magna or Plus mitral valve. Animals were followed with serial transthoracic echocardiography and laboratory studies. At 140 days the sheep underwent cardiac angiography with dobutamine challenge, followed by euthanasia and necropsy. Results: All sheep survived the designated study period. Under echocardiography, the Magna group showed significantly higher mean pressure gradients than the Plus group at 14 days (19.1 ± 5.0 mmHg vs 12.4 ± 4.6 mmHg, p=0.04), but similar mean pressure gradients at 90 days (16.8 ± 12.4 mmHg vs 16.0 ± 11.5 mmHg, p=0.91). Intracardiac catheterization at 140 days showed mean transcatheter mitral valve gradients at baseline, low dobutamine, and high dobutamine challenge were 6.3 mmHg, 7.3 mmHg, and 13.3 mmHg respectively for the Magna group, and 4.1 mmHg, 5.3 mmHg, and 7.7 mmHg respectively for the Plus group. All animals had normal end-organ function and nutrition, and no evidence of hemolysis or coagulopathy, except 2 animals in the Plus group that showed distant emboli. Two Plus group animals had severe valvular calcification; 2 animals in each group had mild valvular calcifications. Conclusions: CEP mitral valves Magna (7300TFX) and Plus (6900P) are appropriate control valves for pre-clinical performance and safety evaluation of novel devices in sheep. Both valves exhibit appropriate transvalvular gradient and hemodynamics, appropriate safety profile, and low grades of pathologic changes in the ovine model. KEYWORD: e-PD-32 The authors do not declare any conflict of interest.

Background Transcatheter aortic valve replacement (TAVR) is an effective therapy in treating high-risk patients suffering from aortic stenosis. Animal models used to evaluate safety and efficacy of TAVR devices prior to clinical use lack a stenotic aortic annulus, a critical impediment to long-term TAVR device evaluation. We sought to create a reproducible model of aortic stenosis using a modified aortic annuloplasty (MAA) procedure in sheep, followed by deployment and long-term evaluation of TAVR devices using this model. Methods Twelve sheep underwent the MAA procedure and were recovered. Transthoracic echocardiography (TTE) was used to monitor changes in the aortic annulus in the postoperative period. At 60 days post-MAA, Test group animals were anesthetized for TAVR insertion and Control animals underwent a necropsy. Test animals were recovered following TAVR insertion and observed for a postoperative period of 140 days. Results Twelve sheep survived the annuloplasty procedure and the 60-day recovery period. Gross examination of seven Control group animals revealed the implanted annuloplasty ring segments formed hard protrusions into the aortic annulus. Five sheep in the Test group underwent successful deployment of Abbott's experimental TAVR device without evidence of migration. Examination at 140 days post-TAVR insertion showed all devices tightly anchored within the modified aortic annulus. Conclusions The MAA procedure creates stenotic segments in the aortic annulus with adequate rigidity for anchorage and long-term evaluation of TAVR devices. This represents the first model that successfully mimics human aortic stenosis and provides a clinically relevant TAVR deployment platform for long-term evaluation in sheep.

Innate immune complement activation may contribute to sickle cell disease (SCD) pathogenesis. Ischemia‐reperfusion physiology is a key component of the inflammatory and vaso‐occlusive milieu in SCD and is associated with complement activation. C5a is an anaphylatoxin, a potent pro‐inflammatory mediator that can activate leukocytes, platelets, and endothelial cells, all of which play a role in vaso‐occlusion. We hypothesize that hypoxia‐reoxygenation (H/R) in SCD mice activates complement, promoting inflammation and vaso‐occlusion. At baseline and after H/R, sickle Townes‐SS mice had increased C3 activation fragments and C5b‐9 deposition in kidneys, livers and lungs and alternative pathway Bb fragments in plasma compared to control AA‐mice. Activated complement promoted vaso‐occlusion (microvascular stasis) in SS‐mice; infusion of zymosan‐activated, but not heat‐inactivated serum, induced substantial vaso‐occlusion in the skin venules of SS‐mice. Infusion of recombinant C5a induced stasis in SS, but not AA‐mice that was blocked by anti‐C5a receptor (C5aR) IgG. C5a‐mediated stasis was accompanied by inflammatory responses in SS‐mice including NF‐κB activation and increased expression of TLR4 and adhesion molecules VCAM‐1, ICAM‐1, and E‐selectin in the liver. Anti‐C5aR IgG blocked these inflammatory responses. Also, C5a rapidly up‐regulated Weibel‐Palade body P‐selectin and von Willebrand factor on the surface of human umbilical vein endothelial cells in vitro and on vascular endothelium in vivo. In SS‐mice, a blocking antibody to P‐selectin inhibited C5a‐induced stasis. Similarly, an antibody to C5 that blocks murine C5 cleavage or an antibody that blocks C5aR inhibited H/R‐induced stasis in SS‐mice. These results suggest that inhibition of C5a may be beneficial in SCD.

BACKGROUND AND AIM OF THE STUDY:Xenograft conduits have been used successfully to repair congenital heart defects, but are prone to failure over time. Hence, in order to improve patient outcomes, better xenografts are being developed. When evaluating a conduit's performance and safety it must first be compared against a clinically available control in a large animal model. The study aim was to evaluate a clinically available xenograft conduit used in right ventricular outflow tract (RVOT) reconstruction in a sheep model.METHODS:RVOT reconstruction was performed in 13 adult and juvenile sheep, using the Medtronic Hancock® Bioprosthetic Valved Conduit (Hancock conduit). The method had previously been used on patients, and a newly modified variant termed 'RVOT Extraction' was employed to facilitate the surgical procedure. Animals were monitored over predetermined terms of 70 to 140 days. Serial transthoracic echocardiography, intracardiac pressure measurements and angiography were performed. On study completion the animals were euthanized and necropsies performed.RESULTS:Two animals died prior to their designated study term due to severe valvular stenosis and distal conduit narrowing, respectively. Thus, 11 animals survived the study term, with few or no complications. Generally, maximal and mean transvalvular pressure gradients across the implanted conduits were increased throughout the postoperative course. Among 11 full-term animals, seven conduits were patent with mild or no pseudointimal proliferation and with flexible leaflets maintaining the hemodynamic integrity of the valve.CONCLUSIONS:RVOT reconstruction using the Hancock conduit was shown to be successful in sheep, with durable and efficient performances. With its extensive clinical use in patients, and ability for long-term use in sheep (as described in the present study) it can be concluded that the Hancock conduit is an excellent control device for the evaluation of new xenografts in future preclinical studies.

Abstract Introduction Innate immune complement activation may contribute to sickle cell disease (SCD) pathogenesis. The alternative complement pathway is abnormally activated in SCD and is additionally activated by phosphatidylserine (PS) on the outer leaflet of SS-red blood cells (SS-RBC). PS on the surface of SS-RBC and activated platelets accelerates the assembly of prothrombinase complexes leading to generation of thrombin, which can cleave circulating C5 protein into two biologically active fragments, C5a and C5b. C5a is an anaphylatoxin, a potent pro-inflammatory mediator, that can activate leukocytes, platelets, and endothelial cells, all of which play a role in vaso-occlusion (VO). Active C5b fragments stimulate the formation of membrane attack complexes (MAC) on SS-RBC that increases their susceptibility to lysis. We hypothesize that complement activation on the surface of SS-RBC may stimulate VO and RBC turnover in SCD. Methods and Results Whole blood from Townes-AA, -AS, and -SS mice (n=4) was collected in EDTA and RBC were immunostained with erythroid specific anti-Ter119 (Ly-76) IgG conjugated to PE-Cy7, anti-C5b-9 (MAC) IgG conjugated to Alexa Fluor 647, and anti-C3 (also specific for activation fragments C3b, iC3b, C3d and C3dg) IgG conjugated to PE. The percentages of Ter119-positive RBC that were positive for MAC were 6.3%, 6.5%, and 26.0% for AA-, AS-, and SS-RBCs, respectively (p<0.01 SS vs. AA and AS) suggesting enhanced C5 activation in Townes-SS mice. However, EDTA plasma C5a levels measured by ELISA were not significantly different between Townes-AA, -AS and -SS mice. C3 activation fragments were also found on a subset of MAC positive RBC. We used a mouse model of hypoxia/reoxygenation (H/R)-induced stasis to investigate the role of complement in VO. Townes-SS mice with implanted dorsal skin-fold chambers were infused with 30 µg of either anti-C5 IgG mAb BB5.1 (which blocks murine C5 cleavage ) (n=4), annexin V (n=2), or with isotype control IgG (n=4) 30 minutes prior to H/R (1 hour of hypoxia at 7% O2, followed by reoxygenation for 4 hours in room air). Percent microvascular stasis (% non-flowing venules) in the subcutaneous venules in the dorsal skin fold chamber window was measured using intravital microscopy at 1 and 4 hours post-hypoxia. Percent stasis was significantly lower in the Townes-SS mice receiving anti-C5 IgG or annexin V than the mice receiving the isotype control IgG at 1 and 4 hours post-hypoxia (Figure 1, means ± SD, *p<0.01 anti-C5 IgG or annexin V vs. control IgG). This finding implies that H/R may acutely activate C5 , thus leading to stasis. Initial studies suggest increased plasma C5a levels in Towne-SS mice treated with isotype control IgG compared to mice treated with anti-C5 IgG in response to H/R. Conclusions These results demonstrate an increased percentage of SS-RBC expressing MAC on their surface and inhibition of H/R-induced stasis with anti-C5 IgG. However, circulating C5a levels were not different between untreated Townes-AA, -AS, or -SS mice suggesting that C5a is not chronically elevated in SCD. Rather, the generation of C5a may occur locally and in the acute setting. Current studies underway are examining the effects of anti-C5 IgG on MAC deposition and RBC half-lives; the mechanism(s) of complement activation in SCD mice (classical, alternative, tic-over, etc); the ability of recombinant murine C5a to induce stasis; and complement activation markers on RBC and in plasma from SCD patients and controls. Disclosures Chen: Imara: Research Funding. Belcher:Imara: Research Funding; CSL-Behring: Research Funding. Vercellotti:Imara: Research Funding; CSL-Behring: Research Funding.

Endothelial cell activation and injury by the terminal pathway of complement is important in various pathobiological processes, including xenograft rejection. Protection against injury by human complement can be induced in porcine endothelial cells (ECs) with IL‐4 and IL‐13 through metabolic activation. However, despite this resistance, the complement‐treated ECs were found to lose membrane permeability control assessed with the small molecule calcein. Therefore, to define the apparent discrepancy of permeability changes vis‐à‐vis the protection from killing, we now investigated whether IL‐4 and IL‐13 influence the release of the large cytoplasmic protein lactate dehydrogenase (LDH) in ECs incubated with complement or the pore‐forming protein melittin. Primary cultures of ECs were pre‐treated with IL‐4 or IL‐13 and then incubated with human serum as source of antibody and complement or melittin. Cell death was assessed using neutral red. Membrane permeability was quantitated measuring LDH release. We found that IL‐4‐/IL‐13‐induced protection of ECs from killing by complement or melittin despite loss of LDH in amounts similar to control ECs. However, the cytokine‐treated ECs that were protected from killing rapidly regained effective control of membrane permeability. Moreover, the viability of the protected ECs was maintained for at least 2 days. We conclude that the protection induced by IL‐4/IL‐13 in ECs against lethal attack by complement or melittin is effective and durable despite severe initial impairment of membrane permeability. The metabolic changes responsible for protection allow the cells to repair the membrane injury caused by complement or melittin.

Injury to endothelial cells (ECs) often results in cell retraction and gap formation. When caused by antigen aggregation or complement, this injury can be prevented by pretreatment of the ECs with IL-4, suggesting that IL-4 modifies the intercellular junction. Therefore, we investigated the effects of IL-4 on expression of intercellular junction proteins and whether such effects are required for IL-4-induced resistance of ECs against complement-mediated injury. We found that IL-4 induces upregulation of the junction protein claudin-5 in porcine ECs through activation of Jak/STAT6 and phosphorylation and translocation of FoxO1 from the nucleus to the cytoplasm. Increased claudin-5 expression resulted in increased transmembrane electrical resistance of the endothelial monolayer and participated in IL-4-induced protection of the ECs from complement injury. Down-regulation of FoxO1 using siRNA by itself caused up-regulation of claudin-5 expression and partial protection from cytotoxicity. This protection was enhanced by stimulation with IL-4. We previously reported that increased phospholipid synthesis and mitochondrial protection were required for IL-4-induced resistance of ECs against complement injury and now we demonstrate a contribution of claudin-5 expression in IL-4-induced protection.

Dalmasso AP. On the intersections of basic and applied research in xenotransplantation. Xenotransplantation 2012; 19: 137–143. © 2012 John Wiley & Sons A/S.Abstract: I am very grateful to the Council and members of the International Xenotransplantation Association for this Honorary Membership. In accepting this prestigious award, I pay tribute to my mentors Antonio Oriol i Anguera, Carlos Martinez, Robert A. Good, and Hans Müller‐Eberhard for their guidance and friendship as I was beginning my travels in biomedical research. I also thank the many gifted collaborators, students, and technical personnel, as well as the agencies and taxpayers, who funded our research and made our scientific contributions possible. Here I briefly mention some of these contributions, including early work on the immunobiology of the thymus, my short incursion in the immunology of Chagas disease, and what have been the dominant themes of my career: the mechanisms of complement injury, the role of complement in pathophysiology, and induction of cytoprotection in the vascular endothelium. I emphasize our contributions on the role of complement as related to understanding and overcoming xenograft injury, a work that has been personally very rewarding. Now it is exciting to see that the field of xenotransplantation research is moving forward vigorously, a time of great optimism suggesting that many potential clinical applications of xenotransplantation will come to fruition in the near future.

Black SM, Benson BA, Idossa D, Vercellotti GM, Dalmasso AP. Protection of porcine endothelial cells against apoptosis with interleukin‐4. Xenotransplantation 2011; 18: 343–354. © 2011 John Wiley & Sons A/S.Abstract:Background: Apoptosis is crucial for tissue development and homeostasis, and insufficient apoptosis is pivotal in cancer pathogenesis. Apoptosis may also be important in tissue injury and in this case, it is of interest to induce protection against apoptosis. In organ transplantation, apoptosis has been implicated in acute vascular rejection (AVR); in xenotransplantation, the inducers of apoptosis of relevance in AVR, such as tumor necrosis factor‐α (TNF‐α), also cause endothelial cell (EC) activation. We have previously shown that interleukin (IL)‐4 and IL‐13 induced protection in porcine ECs against activation and apoptosis triggered by TNF‐α. Now we define signaling processes activated by IL‐4 in porcine ECs and mechanisms required for IL‐4‐induced protection against apoptosis.Methods: Porcine aortic ECs were used as primary cultures or as virus‐induced immortalized cells derived from galactosyl transferase‐deficient (Gal−/−) or wild‐type pigs. ECs were stimulated with porcine IL‐4, either extrinsically or transduced with recombinant adenovirus (adeno) IL‐4, and analyzed using immunoblotting. Apoptosis was induced with TNF‐α plus cycloheximide and assessed using neutral red uptake or flow cytometry. The role of various signaling proteins in IL‐4‐induced protection was established using pharmacologic inhibitors and siRNA downregulation of protein expression.Results: IL‐4 induced similar degrees of phosphorylation of STAT6 in all 3 types of ECs, and STAT6 was phosphorylated through Jak3. IL‐4 induced phosphorylation of Bad through Jak3. Stimulation of ECs with IL‐4 caused protection of ECs against apoptosis with an absolute requirement of Jak3/STAT6 activation and major participation of mammalian target of rapamycin complex 2 (mTORC2), Akt, and extracellular signal‐regulated kinase 1/2. IL‐4 caused no increase in EC levels of protective proteins hemoxygenase‐1, inhibitor of apoptosis protein, heat shock protein 70, Bcl‐2, and Bcl‐xL. ECs transduced with adenoIL‐4 exhibited strong and durable protection from apoptosis. Gal−/−ECs were as susceptible to induction of protection with IL‐4 as wild‐type ECs.Conclusions: IL‐4 induces activation of Jak3/STAT6 and phosphorylation of Bad in porcine ECs, ultimately resulting in effective protection of the ECs from apoptosis. Delineation of downstream signals activated by IL‐4 that are required for induction of protection suggests possible sites of intervention to design effective therapeutic agents. This is of interest because substances such as IL‐4 have pleiotropic effects and cannot be used directly due to potential deleterious effects. Inducing resistance to apoptosis in porcine vascular endothelium may be important to facilitate xenograft survival and accommodation.

We have shown previously that cytokines IL-4 and IL-13 induce protection in porcine vascular endothelial cells (EC) against killing by the membrane attack complex (MAC) of human complement. This protection is intrinsic, not due to changes in complement regulatory proteins, and requires activation of Akt and sterol receptor element binding protein-1 (SREBP-1), which regulates fatty acid and phospholipid synthesis. Here we report that, compared to EC incubated in medium, IL-4-treated EC had a profound reduction in complement-mediated ATP loss and in killing assessed by vital dye uptake, but only a slight reduction in permeability disruption measured by calcein release. While controls exposed to complement lost mitochondrial membrane potential and subsequently died, protected EC maintained mitochondrial morphology and membrane potential, and remained alive. SREBP-1 and fatty acid synthase activation were required for protection and fatty acid and phospholipid synthesis, including cardiolipin, were increased after IL-4 stimulation, without increase in cholesterol content or cell proliferation. IL-4 also induced protection of EC from killing by the channel forming protein melittin, similar to protection observed for the MAC. We conclude that IL-4 induced activation of Akt/SREBP-1/lipid biosynthesis in EC, resulting in protection against MAC and melittin, in association with mitochondrial protection.

BACKGROUND:To induce accommodation in the hamster-to-rat cardiac transplantation model, in addition to cyclosporin A (CSA) to inhibit T-cell-mediated graft rejection, cobra venom factor (CVF) is often used to prevent complement-mediated graft rejection. Although it is generally assumed that CVF makes accommodation possible because it inactivates the complement membrane attack complex (MAC), it is not known which complement components must be inactivated and whether complement activation products generated by CVF are also involved in the induction of accommodation. Therefore, to investigate mechanisms by which CVF contributes to accommodation, we studied induction of accommodation of hamster hearts grafted into rats with complement deficiencies of C6; these rats cannot assemble the MAC but, in contrast to CVF, retain in their native state all complement proteins that precede the MAC.METHODS:Golden Syrian hamster hearts were transplanted heterotopically into the abdomen of normocomplementemic and C6-deficient (C6D) PVG rats. Graft rejection was determined by cessation of palpable cardiac contractions. CSA, 10 mg/kg, was administered daily to all rats. Graft survival was compared in rats given CVF (60 U/kg 1-day pre-transplant and 20 U/kg/day for the next 9 days), C6D rats given no CVF, normocomplementemic rats given anti-C6 IgG or non-immune IgG but no CVF, and C6D rats reconstituted with normocomplementemic rat serum. Total complement and C6 serum levels were measured using hemolytic assays in rat peripheral blood.RESULTS:We found that hamster hearts transplanted into C6D rats receiving CSA but no CVF survived long-term, with histology typical of an accommodated heart. The accommodated hamster heart did not reconstitute C6 levels of the C6D recipient rats. Moreover, in normocomplementemic rats given anti-C6 antibodies (abs) to induce partial C6 deficiency, accommodation also developed without administration of CVF. Accommodation of the hamster heart failed to develop in C6D rats whose complement was reconstituted by administration of normocomplementemic rat serum given before and following transplantation.CONCLUSIONS:These studies demonstrate that, in this model, inhibition of MAC-mediated graft rejection is sufficient to allow the development of accommodation. Inactivation of C3 or other complement proteins of the alternate pathway, or the presence of complement-derived biologically active fragments, is not needed for development of accommodation.

Vascular endothelial cells (ECs) can be injured in a variety of pathologic processes that involve activated complement. We reported previously that porcine ECs incubated with exogenous IL-4 or IL-13 are protected from cytotoxicity by human complement and also from apoptosis by TNF-alpha. The resistance to complement consists of an intrinsic mechanism that is lost a few days after cytokine removal. In our current study, we investigated whether transfer of the IL-4 gene into porcine ECs in vitro and into porcine vascular tissues in vivo would induce efficient and durable protection from human complement. We found that ECs transduced with adenoIL-4 or adenoIL-13 exhibited continuous production of the cytokine and prolonged protection from complement-mediated killing. IL-4 also protected ECs from activation: ECs incubated with IL-4 did not develop cell retraction and intercellular gaps upon stimulation with sublytic complement. The endothelium and subendothelium of pig iliac arteries that were transduced with the IL-4 gene were effectively protected from complement-dependent immediate injury after perfusion with human blood. However, after similar perfusion, the endothelium was immediately lost from arteries that were transduced with a control adenovirus. The protection was not due to up-regulation of the complement regulators decay accelerating factor, membrane cofactor protein, and CD59, or to reduced complement activation, but required the participation of Akt. Although our studies model protection in pig-to-primate xenotransplantation, our findings of IL-4 induction of Akt-mediated protection may be more broadly applicable to EC injury as manifested in ischemia-reperfusion, allotransplantation, and various vascular diseases.

Studies in rodent models suggested that complement may play a critical role in susceptibility to airway hyperresponsiveness (AHR) and as a mediator of bronchial obstruction and inflammation in asthma. Complement may participate in susceptibility to asthma because of an intrinsic abnormality in complement activation and generation of C3a, C5a, or other products that affect cellular responses, resulting in T(H)2 predominance and asthma susceptibility. Alternatively, an intrinsic abnormality in the cellular response to complement activation products could determine susceptibility to asthma. In this study, the authors investigated whether complement in patients with atopic asthma versus nonatopic controls possesses an increased propensity to become activated. Despite reports that total complement plasma levels in unchallenged asthmatics are normal, an abnormal sensitivity of complement to activation may exist if an isoform or a polymorphic variant of a complement protein was present and resulted in gain or loss of function without associated changes in total complement levels. Therefore, complement activation was induced in vitro in plasma of asthmatics and controls using activators of the classical, alternative, and lectin pathways and measured C3a, other C3 fragments, and C5a. For each pathway, similar amounts of generated fragments, as well as C3a/C3 and C5a/C5 ratios, were found in asthmatics and controls. Also, similar basal plasma levels of C3a and C5a were found in both groups; however, mannan-binding lectin (MBL) levels were moderately elevated in asthmatics. In conclusion, the results suggest that, in asthmatic patients, complement activation does not exhibit an abnormal sensitivity to activation by any of the known activation pathways.

Cytoprotection of endothelial cells (EC) is important in EC biology and pathophysiology, including graft rejection. Using porcine aortic EC and human complement as an in vitro model of xenotransplantation, we have reported that ligation of EC Galα(1–3)Gal epitopes (αGal) with antibodies or lectins BS-I and IB4 induces EC resistance to injury by complement. However, before the protective response is observed, αGal ligation induces an early, proinflammatory response. Using a similar model, we now investigated whether the early inflammatory response, as well as NF-κB activation, is required for induction of cytoprotection. Despite up-regulation of EC mRNA for many inflammatory cytokines rapidly after BS-I stimulation, recombinant cytokines or conditioned media from EC incubated with BS-I failed to induce protection when used to stimulate EC. While the lectin-induced inflammatory response was markedly reduced by inhibition of NF-κB, the protection from complement and apoptosis was unaffected. The lectins caused up-regulation of mRNA for protective genes A20, porcine inhibitor of apoptosis protein and hemoxygenase-1, which was not modified by NF-κB inhibition. These findings suggest that induction of cytoprotection in porcine EC by αGal ligation results from activation of pathways that are largely independent of those that elicit NF-κB activation and the inflammatory response.