Team:Slovenia/ImplementationIschaemicHeartDisease

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Ischaemic heart disease

We designed a device for the therapy of myocardial ischaemia, composed of microencapsulated mammalian cells that include a genetic bistable toggle switch with a positive feedback loop, where in the first state cells produce anakinra as the anti-inflammatory effector and in the second state they produce a stoichiometric amount of the vascular endothelial growth factor (VEGF) and platelet-derived growth factor B (PDGF-BB) to promote angiogenesis in the damaged tissue.

A pharmacokinetic model demonstrated that implantation of the device into the injured heart tissue results in a high level of anakinra concentration within the affected tissue, while the systemic level of anakinra is negligible, preventing systemic immunosupression.

We demonstrated that the production level of anakinra by engineered cells is sufficient for the therapeutic implementation of microencapsulated cells for this indication.

Ischaemic heart disease

Ischaemic heart disease is characterized by a reduced blood supply to the heart muscle, usually due to coronary artery disease (atherosclerosis of the coronary arteries). Ischaemic heart disease (which includes myocardial infarction, angina pectoris and heart failure when preceded by myocardial infarction) is the leading cause of mortality in most Western countries. Menzin et al. reported a total first-year cost averaged of 32,345 $ and as much as 61% of these costs were due to rehospitalisation (Menzin et al., 2008). Furthermore, survivors of acute myocardial infarction remain at high risk of death in the years after the event (Grothusen et al., 2012).

Current therapy and research

Acute myocardial infarction due to total occlusion of a coronary artery is treated with early reperfusion strategies to minimize the developing ischaemic myocardial damage. Current guidelines recommend interventional treatment via percutaneous transluminal coronary angioplasty (PTCA) of the occluded coronary artery followed by a stent implantation in the setting of acute myocardial infarction. In case of PTCA contraindications, operative revascularization therapy CABG (coronary artery bypass graft surgery) is recommended. However, many studies have shown a negative effect of reperfusion, resulting in additional myocardial injury due to activation of an inflammatory response (Grothusen et al., 2012; Abbate et al., 2008).

Interleukin-1a and b are one of the most important inflammatory cytokines which are responsible for leukocyte chemotaxis, macrophage activation, reactive oxygen species formation, endothelial dysfunction, and cardiomyocyte apoptosis. Thus, inhibition of IL-1 receptor complex activation can present a new important treatment target (Grothusen et al., 2012). Furthermore, recent studies have shown the cardioprotective properties of anakinra, which is a nonglycosylated, human recombinant competitive inhibitor of IL-1a and b signaling through binding to the IL-1 receptor complex (Abbate et al., 2008).

There is also experimental evidence of the important regenerating role of vascular endothelial growth factor (VEGF) after acute myocardial infarction (Banfi et al., 2012; Zhang et al., 2008). After binding to its receptor VEGFR1 and VEGFR2 it supports angiogenesis, inhibits endothelial cell apoptosis, promotes endothelial cell proliferation, and restores heart function. Although therapeutic angiogenesis by delivery of vascular growth factors is an attractive strategy, many clinical trials have thus far failed to show efficiency. One of the most likely explanations for this discrepancy is that VEGF induces growth of dysfunctional vessels, if expressed outside of a narrow dosage window (Banfi et al., 2012). Banfi et al. confirmed the hypothesis that co-delivery of platelet-derived growth factor-BB (PDGF-BB), which recruits pericytes, induced normal angiogenesis in skeletal muscle irrespective of VEGF levels. It was also shown that coexpression of VEGF and PDGF-BB encoded by separate vectors in different cells or in the same cells only partially corrected aberrant angiogenesis. In marked contrast, coexpression of both factors in every cell at a fixed relative level via a single bicistronic vector led to robust, uniformly normal angiogenesis, even when VEGF expression was high and heterogeneous. Secondly, a challenge has been that with the conventional gene transfer vectors, the growth factor concentration in target tissues had not reached sufficient levels or had not persisted long enough for triggering relevant vascular growth (Zhang et al., 2008). Cell transplantation strategies emerged as a promising approach to overcome this issue.

Zhang et al. reported the first transplantation of microencapsulated engineered xenogeneic CHO cells in post-infarction myocardium, which showed that the supplementation of VEGF from implanted xenogeneic cells could foster the formation of arterial collaterals, improve myocardial perfusion and thus also promote the regeneration of damaged myocardium augmented angiogenesis and improved heart function (Zhang et al., 2008).

Implementation of the microencapsulated engineered cellular device for the therapy of ischaemic heart disease

With our biological delivery system for biopharmaceuticals, we aim to improve the therapy of ischaemic heart disease by delivering multiple biopharmaceuticals in different time periods as controlled by the physician. The encapsulated cells would be locally administered with intracardial injections via ventriculography, or intraoperatively in the proximity of the myocardial damage (Figure 1). In the first phase, after myocardial damage has occurred and reperfusion injury is expected, production of anakinra could halt the inflammatory response. In the second phase, the therapy would switch to the production of VEGF and PDGF-BB at stoichiometric concentrations, which could enhance the production of normal, robust vessels (Figure 2). After the therapeutic effect is reached, we could discontinue the synthesis of growth factors through the ability to control the viability of encapsulated allogenic cells. This could prevent the negative side effects of over-production of vascular growth factors (haemangioma formation) and contribute a great deal to biosafety. Because of the local application, we anticipated that systemic effects of anakinra, VEGF and PDGF would be negligible but we tested this by a pharmacokinetic model. This strategy of treatment could prevent the development of chronic heart failure, which is one of the commonest late complications after myocardial infarction.

Results

Pharmacokinetic modeling of the distribution of anakinra in the local antiinflammatory therapy

We prepared a pharmacokinetic model for the local therapy of ischaemic heart disease with anakinra. This model (described in details in the Modeling section) demonstrates achievement of the local therapeutic concentrations of anakinra, while the systemic concentration remains negligible in contrast to systemic application of anti-inflammatory therapy. The serum half life of anakinra is very low so it needs to be administered daily for a systemic application, but implementation of encapsulated cells with a constant production rate of anakinra would circumvent this.

Figure 3. Distribution of anakinra in different tissues and organs based on the pharmacokinetic model of the local therapy of ischaemic heart disease by microencapsulated engineered cells.

Detection of biological activity of produced anakinra

IL-1β signals through binding of IL-1 receptor type 1 (IL-1R1) and IL-1 receptor accessory protein (IL-1RAcP), which leads to a juxtaposition of the intracellular TIR domains of IL-1R1 and IL-1RAcP. The activation of the receptor complex triggers intracellular signaling which results in the activation of the transcription factor NF-κB and the mitogen-activated protein kinase (MAPK) pathways. Anakinra (IL-1 receptor antagonist; IL-1Ra) is a competitive inhibitor that prevents activation of the IL-1 receptor complex (Wang et al., 2010).

We wanted to test the biological activity of anakinra produced in HEK293T. We designed the experiment where the inhibition of IL-1β signaling by anakinra was observed. HEK293T cells were transfected with NF-κB-inducible firefly luciferase reporter plasmid and constitutive Renilla luciferase plasmid for normalization. Transfected cells were preincubated with supernatant from anakinra-producing cells or with recombinant anakinra (rIL-1Ra). These cells were then stimulated with IL-1β and inhibition of NF-κB signaling pathway by anakinra was observed with double luciferase assay (Figure 4). Results show almost complete inhibition of IL-1β signaling with recombinant anakinra as well as with supernatant from anakinra-producing cells.

Figure 4. Inhibition of IL-1β signaling by anakinra. HEK293T cells, transfected with reporter plasmid, were preincubated with supernatant of HEK293T cells producing anakinra under the control of a constitutive promoter or with recombinant anakinra (rIL-1Ra) (500 ng/mL). These cells were then stimulated with IL-1β (10 ng/mL) and inhibition of NF-κB signaling pathway by anakinra was observed with double luciferase assay.

We have calculated the therapeutic concentration of anakinra to be 25 ng/mL, which we derived from the anakinra IC_50 value (Dahlén et al., 2008). Our pharmacokinetic model of microencapsulated cell therapy predicted we would need production of approximately 30 µg per day in the target tissue. Considering that in our experiments each cell produced around 1*10^-6 µg per day and that number of cells per capsule is 3000-15000 we calculated that we would need to implant between 2000-9000 capsules into a patch of ischaemic tissue, which would represent a volume of 15 to 75uL. Those estimates are very rough and would have to be tested experimentally in the tissue but provide the basis for judging the feasibility of this approach. Additional indication may be also provided by the successful application of the local gene therapy of myocarditis (Lim et al., 2002, Suzuki et al., 2001)

References

Abbate, A., Salloum, F.N., Vecile, E., Das, A., Hoke, N.N., Straino, S., Biondi-Zoccai, G.G., Houser, J.E., Qureshi, I.Z., Ownby, E.D., Gustini, E., Biasucci, L.M., Severino, A., Capogrossi, M.C., Vetrovec, G.W., Crea, F., Baldi, A., Kukreja, R.C., and Dobrina, A. (2008) Anakinra, a recombinant human Interleukin-1 receptor antagonist, inhibits apoptosis in experimental acute myocardial infarction. Circulation 17, 2670-2683.

Banfi, A., von Degenfeld, G., Gianni-Barrera, R., Reginato, S., Merchant, M.J., McDonald, D.M., and Blau, H.M. (2012) Therapeutic angiogenesis due to balanced single-vector delivery of VEGF and PDGF-BB. FASEB J. 26, 2486-2497.

Dahlén, E., Barchan, K., Herrlander, D., Höjman, P., Karlsson, M., Ljung, L., Andersson, M., et al. 2008. Development of Interleukin-1 Receptor Antagonist Mutants with Enhanced Antagonistic Activity In Vitro and Improved Therapeutic Efficacy in Collagen-Induced Arthritis.J Immunotoxicol 5, 189-99.

Grothusen, C., Hagemann, A., Attmann, T., Braesen, J., Broch, O., Cremer ,J., and Schoettler, J. (2012) Impact of an interleukin-1 receptor antagonist and erythropoietin onexperimental myocardial ischemia/reperfusion injury. Scientific World Journal 737585. Epub 2012 May 2

Lim BK, Choe SC, Shin JO, Ho SH, Kim JM, Yu SS, Kim S, Jeon ES. (2002) Local expression of interleukin-1 receptor antagonist by plasmid DNA improves mortality and decreases myocardial inflammation in experimental coxsackieviral myocarditis.Circulation 105, 1278-81.

Menzin, J., Wygant, G., Hauch, O., Jackel, J., and Friedman, M. (2008) One-year costs of ischemic heart disease among patients with acute coronary syndromes: findings from a multi-employer claims database. Curr. Med. Res. Opin. 24, 461-468.

Suzuki K, Murtuza B, Smolenski RT, Sammut IA, Suzuki N, Kaneda Y, Yacoub MH. (2001) Overexpression of interleukin-1 receptor antagonist provides cardioprotection against ischemia-reperfusion injury associated with reduction in apoptosis. Circulation 104, I308-I3.

Wang D., Zhang S., Li L., Xi Liu, Mei K. and Wang X. (2010) Structural insights into the assembly and activation of IL-1b with its receptors. Nat. immunol 11, 10, 905-912.

Zhang, H., Zhu, S.J., Wang, W., Wei, Y.J., and Hu, S.S. (2008) Transplantation of microencapsulated genetically modified xenogeneic cells augments angiogenesis and improves heart function. Gene Ther. 15, 40-48.

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