Heme Oxygenase-1 Gene Therapy Provides Cardioprotection Via Control of Post-Ischemic Inflammation: An Experimental Study in a Pre-Clinical Pig Model.

BACKGROUND: Heme oxygenase-1 (HO-1) is an inducible stress-responsive enzyme converting heme to bilirubin, carbon monoxide, and free iron, which exerts anti-inflammatory and antiapoptotic effects. Although efficient cardioprotection after HO-1 overexpression has been reported in rodents, its role in attenuating post-ischemic inflammation is unclear. OBJECTIVES: This study assessed the efficacy of recombinant adenoassociated virus (rAAV)-encoding human heme oxygenase-1 (hHO-1) in attenuating post-ischemic inflammation in a murine and a porcine ischemia/reperfusion model. METHODS: Murine ischemia was induced by 45 min of left anterior descending occlusion, followed by 24 h of reperfusion and functional as well as fluorescent-activated cell sorting analysis. Porcine hearts were subjected to 60 min of ischemia and 24h of reperfusion before hemodynamic and histologic analyses were performed. RESULTS: Human microvascular endothelial cells transfected with hHO-1 displayed an attenuated interleukin-6 and intercellular adhesion molecule 1 expression, resulting in reduced monocytic THP-1 cell recruitment in vitro. In murine left anterior descending occlusion and reperfusion, the post-ischemic influx of CD45(+) leukocytes, Ly-6G(+) neutrophils, and Ly-6C(high) monocytes was further exacerbated in HO-1-deficient hearts and reversed by rAAV.hHO-1 treatment. Conversely, in our porcine model of ischemia, the post-ischemic influx of myeloperoxidase-positive neutrophils and CD14(+) monocytes was reduced by 49% and 87% after rAAV.hHO-1 transduction, similar to hHO-1 transgenic pigs. Functionally, rAAV.hHO-1 and hHO-1 transgenic left ventricles displayed a smaller loss of ejection fraction than control animals. CONCLUSIONS: Whereas HO-1 deficiency exacerbates post-ischemic cardiac inflammation in mice, hHO-1 gene therapy attenuates inflammation after ischemia and reperfusion in murine and porcine hearts. Regional hHO-1 gene therapy provides cardioprotection in a pre-clinical porcine ischemia/reperfusion model.

SDF-1 fused to a fractalkine stalk and a GPI anchor enables functional neovascularization.

The facilitated recruitment of vascular progenitor cells (VPCs) to ischemic areas might be a therapeutic target for neovascularization and repair. However, efficient and directed attraction of VPCs remains a major challenge in clinical application. To enhance VPC homing, we developed a fusion protein (S1FG), based on the biology of stroma-derived factor-1/CXCL12 and the mucin backbone taken from fractalkine/CXCL12. A GPI-anchor was included to link the fusion-protein to the cell surface. HUVECs transfected with S1FG were capable of increasing firm adhesion of CXCR4+-mononuclear cells (THP-1) under shear stress conditions in vitro. In an in vivo rabbit model of chronic hind limb ischemia, local S1FG application enhanced the recruitment of adoptively transferred embryonic EPCs (eEPCs) to the ischemic muscles 2.5-fold. S1FG combined with eEPC(low) (2 × 10(6)) yielded similar capillary growth as eEPC(high) (5 × 10(6)) alone. Compared to controls, collateral formation was increased in the S1FG eEPC(low) group, but not the eEPC(high) group without S1FG, whereas perfusion was found enhanced in both groups. In addition, S1FG also increased collateral formation and flow when combined with AMD3100 treatment, to increase circulating levels of endogenous VPC. These data demonstrate that the fusion protein S1FG is capable of enhancing the recruitment of exogenously applied or endogenously mobilized progenitor cells to sites of injury. Recombinant versions of S1FG applied via catheters in combination with progenitor cell mobilization may be useful in the treatment of chronic ischemic syndromes requiring improved perfusion.

Angiogenetic potential of Ad2/Hif-1α/VP16 after regional application in a preclinical pig model of chronic ischemia.

UNLABELLED: Hif-1α, a master regulator of ischemia-responsive gene induction, controls pro-angiogenic gene expression of VEGF-A, flt-1, IGF-1 and erythropoietin, rendering its overexpression an attractive tool for therapeutic neovascularization. Utilizing an adenoviral vector system, we investigated the efficacy of selective pressure-regulated venous retroinfusion of an enhanced Hif-1α mutant (Hif-1α/VP16) in a randomized investigator-blinded study. METHODS: Pigs were subjected to percutaneous implantation of a reduction-stent into the circumflex artery, leading to progressive stenosis and complete occlusion at day 28. Selective pressure-regulated retroinfusion of the great cardiac vein was performed at day 28 for regional delivery of either saline or empty vector or Ad2/Hif-1α/VP16. Collateral growth and global myocardial function were obtained by fluoroscopy, whereas regional blood flow and regional myocardial function were assessed by fluorescent microsphere analysis and sonomicrometry, respectively. Capillary density in the ischemic myocardium was analyzed by PECAM-1 staining. RESULTS: Compared to saline and Ad empty vector controls, overexpression of Hif-1α in the ischemic region induced an increase of small (capillary) and large (collateral) vessels, resulting in an improved perfusion of the ischemic myocardium. Concomitantly, an ischemia induced loss of myocardial function (hibernating myocardium) was resolved only after transfection with the Hif 1-α transgene, but not the empty vector or saline control. CONCLUSION: Retroinfusion of Ad2/Hif-1α/VP16, combining a master pro-angiogenic protein with regional myocardial application, may offer an efficient approach to cardiac gene therapy of chronic ischemic cardiomyopathy.

Cotransfection of vascular endothelial growth factor-A and platelet-derived growth factor-B via recombinant adeno-associated virus resolves chronic ischemic malperfusion role of vessel maturation.

OBJECTIVES: We set out to investigate the ability of cardiotropic adeno-associated viral vector (AAV2.9 = recombinant adeno-associated virus [rAAV]) to induce prolonged expression of vascular endothelial growth factor (VEGF)-A and platelet-derived growth factor (PDGF)-B in a rabbit hindlimb ischemia model and a pig model of hibernating myocardium. BACKGROUND: Gene therapy to induce angiogenesis and arteriogenesis has produced mixed results. However, long-acting viruses, such as rAAV, as well as combined induction of angiogenesis and vessel maturation might extend the therapeutic potential. METHODS: In rabbits, 0.5 x 10(11) particles rAAV.VEGF-A with or without 1 x 10(12) particles rAAV.PDGF-B were retroinfused at day 7 after femoral artery excision. At days 7 and 35, collateral counts and perfusion were determined, each value given as the day 35/day 7 ratio. Capillary-to-muscle fiber ratio was determined at day 35. In pigs, implantation of a reduction stent graft into the circumflex artery led to complete occlusion at day 28. At this time point, retroinfusion of rAAV.VEGF-A (1 x 10(13) particles), rAAV.VEGF-A/PDGF-B (2 x 10(12) and 4 x 10(12) particles, respectively) or mock transfection was performed. Ejection fraction and left ventricular end-diastolic pressure were assessed at days 28 and 56. RESULTS: In rabbits, rAAV.VEGF-A strongly induced angiogenesis (capillary-to-muscle fiber ratio; 1.67 +/- 0.09 vs. 1.32 +/- 0.11 in rAAV.LacZ-treated limbs, p < 0.05), but not collateral growth (125 +/- 7% vs. 106 +/- 7%, p = NS) or perfusion (136 +/- 12% vs. 107 +/- 9%, p = NS). With VEGF-A/PDGF-B cotransfection, collateral growth increased to 146 +/- 9%, perfusion to 163 +/- 8% of the respective day 7 value (p < 0.05). In the pig model, retroinfusion of rAAV.VEGF-A/PDGF-B increased regional myocardial blood flow reserve from 101 +/- 4% (rAAV.Mock) to 129 +/- 8% (p < 0.05), based on collateral growth (3.2 +/- 0.3 in rAAV.Mock vs. 9.0 +/- 0.4 in rAAV.VEGF-A/PDGF-B, p < 0.05), whereas rAAV.VEGF-A did not alter flow reserve (112 +/- 7%) or collateral count (5.2 +/- 0.7). rAAV.VEGF-A/PDGF-B improved ejection fraction (55 +/- 5% vs. 34 +/- 3% in rAAV.Mock, p < 0.05) unlike rAAV.VEGF-A (37 +/- 2%). CONCLUSIONS: Retroinfusion of rAAV.VEGF-A alone induces angiogenesis, but fails to enhance collateralization and perfusion, unless PDGF-B is cotransfected. In addition to neovascularization, rAAV.VEGF-A/PDGF-B improves regional and global myocardial function in hibernating myocardium.

NF kappaB activation in embryonic endothelial progenitor cells enhances neovascularization via PSGL-1 mediated recruitment: novel role for LL37.

Embryonal endothelial progenitor cells (eEPCs) are capable of inducing therapeutic angiogenesis in a chronic hind limb model. However, the proportion of eEPCs recruited to the ischemic tissue appears to be a limiting step for the induction of cell-based therapeutic neovascularization. In the present study, we primed eEPCs with the human cathelicidin LL37 (hCAP-18) ex vivo to selectively enhance the eEPC-dependent gain of perfusion in vivo and elucidated the mechanism of action of LL37 on eEPCs. Seven days after femoral artery excision, 5 x 10(6) eEPCs (wt, wild type; p65t, transiently p65 transient; p65s, stable p65-transfected; LL37-eEPCs, LL37 peptide preincubated) were retroinfused into the anterior tibial vein. Recruitment of diI-labeled eEPCs in the ischemic gastrocnemic muscle was investigated 2 days later, whereas collateral growth and perfusion score (obtained by fluorescent microspheres) were assessed at day 7 and day 35 and are given as percentage of day 7 level. Capillary/muscle fiber ratio in the ischemic lower limb was obtained at day 35. Embryonic EPC recruitment in vitro and in vivo was found elevated after LL37 and p65t pretreatment, but not in p65s-eEPCs displaying increased IkappaBalpha or after LL37 in IkappaB-DN overexpressing eEPCs. Using LL37- and p65t-eEPCs, collateral growth (181 +/- 10% and 165 +/- 8%, respectively) surpassed that of wt-eEPCs (135 +/- 7%), increasing perfusion ratio (208 +/- 20% and 210 +/- 17% vs. 142 +/- 12% in wt-eEPCs, respectively), whereas p65s-eEPCs exerted no additive effect (collateral growth 130 +/- 8%; perfusion ratio 155 +/- 15%). Moreover, p65t-eEPC-induced neovascularization was abrogated by blocking antibodies against E-selectin and P-selectin glycoprotein ligand-1 (PSGL-1). We conclude that NF kappaB activation by LL37 or transient p65-transfection increases functionally relevant eEPC recruitment to ischemic muscle tissue via induction of PSGL-1 and E-selectin.

Recombinant adeno-associated virus-based gene transfer of cathelicidin induces therapeutic neovascularization preferentially via potent collateral growth.

Therapeutic neovascularization is a concept well validated in animal models, however, without clear-cut success in clinical studies. To achieve prolonged transgene expression, recombinant adeno-associated virus (rAAV) was used in a chronic ischemic hind-limb model and the human antimicrobial peptide cathelicidin (LL-37/hCAP-18) was used as proangiogenic factor. Seven days after femoral artery excision, 0.5 x 10(11) rAAV particles encoding for green fluorescent protein (rAAV.GFP), cathelicidin (rAAV.cath), or vascular endothelial growth factor A (rAAV.VEGF-A) were retroinfused into the anterior tibial vein of rabbits (n = 5 per group). In addition, one rAAV.cath-treated group obtained a constant infusion with the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin into the ischemic tissue starting on day 7. On day 7 and day 35 angiography of both hind limbs was performed for collateral quantification and frame count score (cinedensitometry). Capillary-to-muscle fiber ratios were obtained on day 35. Compared with controls, application of rAAV.cath induced a gain of perfusion (153 +/- 12 vs. 107 + 9% of day 7 controls) via increased collateral growth (length index, 161 +/- 14 vs. 97 +/- 9%, controls), but no significant capillary growth (1.16 +/- 0.09 vs. 0.99 +/- 0.08, controls). Wortmannin application completely abolished the effects of rAAV.cath, indicating the involvement of the PI3K signal pathway. In conclusion, rAAV-mediated cathelicidin expression is capable of inducing functionally relevant neovascularization, preferentially by collateral growth. The rAAV-based vectors as long-expressing vector expression systems and cathelicidin as proangiogenic factor provide a promising new combination in the treatment of peripheral artery disease.

Thymosin beta4 is an essential paracrine factor of embryonic endothelial progenitor cell-mediated cardioprotection.

BACKGROUND: Prolonged myocardial ischemia results in cardiomyocyte loss despite successful revascularization. We have reported that retrograde application of embryonic endothelial progenitor cells (eEPCs) provides rapid paracrine protection against ischemia-reperfusion injury. Here, we investigated the role of thymosin beta4 (Tbeta4) as a mediator of eEPC-mediated cardioprotection. METHODS AND RESULTS: In vitro, neonatal rat cardiomyocytes were subjected to hypoxia-reoxygenation in the absence or presence of eEPCs with or without Tbeta4 short hairpin RNA (shRNA) transfection. In vivo, pigs (n=9 per group) underwent percutaneous left anterior descending artery occlusion for 60 minutes on day 1. After 55 minutes of ischemia, control eEPCs (5x10(6) cells) or cells transfected with Tbeta4 shRNA when indicated or 15 mg Tbeta4 alone were retroinfused into the anterior interventricular vein. Segmental endocardial shortening in the infarct zone at 150-bpm atrial pacing, infarct size (triphenyl tetrazolium chloride viability and methylene blue exclusion), and inflammatory cell influx (myeloperoxidase activity) were determined 24 hours later. Survival of neonatal rat cardiomyocytes increased from 32+/-4% to 90+/-2% after eEPC application, an effect sensitive to shRNA transfection compared with Tbeta4 (45+/-7%). In vivo, infarct size decreased with eEPC application (38+/-4% versus 54+/-4% of area at risk; P<0.01), an effect abolished by Tbeta4 shRNA (62+/-3%). Segmental subendocardial shortening improved after eEPC treatment (22+/-3% versus -3+/-4% of control area) unless Tbeta4 shRNA was transfected (-6+/-4%). Retroinfusion of Tbeta4 mimicked eEPC application (infarct size, 37+/-3%; segmental endocardial shortening, 34+/-7%). Myeloperoxidase activity (3323+/-388 U/mg in controls) was decreased by eEPCs (1996+/-546 U/mg) or Tbeta4 alone (1455+/-197 U/mg) but not Tbeta4 shRNA-treated eEPCs (5449+/-829 U/mg). CONCLUSIONS: Our findings show that short-term cardioprotection derived by regional application of eEPCs can be attributed, at least in part, to Tbeta4.

Endothelial nitric oxide synthase overexpression provides a functionally relevant angiogenic switch in hibernating pig myocardium.

OBJECTIVES: We investigated whether retroinfusion of liposomal endothelial nitric oxide synthase (eNOS) S1177D complementary deoxyribonucleic acid (cDNA) would affect neovascularization and function of the ischemic myocardium. BACKGROUND: Recently, we demonstrated the feasibility of liposomal eNOS cDNA transfection via retroinfusion in a model of acute myocardial ischemia/reperfusion. In the present study, we used this approach to target a phosphomimetic eNOS construct (eNOS S1177D) into chronic ischemic myocardium in a pig model of hibernation. METHODS: Pigs (n = 6/group) were subjected to percutaneous implantation of a reduction stent graft into the left anterior descending artery (LAD), inducing total occlusion within 28 days. At day 28, retroinfusion of saline solution containing liposomal green fluorescent protein or eNOS S1177D cDNA (1.5 mg/animal, 2 x 10 min) was performed. Furthermore, L-nitroarginine-methylester (L-NAME) was applied orally from day 28, where indicated. At day 28 and day 49, fluorescent microspheres were injected into the left atrium for perfusion analysis. Regional functional reserve (at atrial pacing 140/min) was assessed at day 49 by subendocardial segment shortening (SES) (sonomicrometry, percent of ramus circumflexus region). RESULTS: The eNOS S1177D overexpression increased endothelial cell proliferation as well as capillary and collateral growth at day 49. Concomitantly, eNOS S1177D overexpression enhanced regional myocardial perfusion from 62 +/- 4% (control) to 77 +/- 3% of circumflex coronary artery-perfused myocardium, unless L-NAME was co-applied (69 +/- 5%). Similarly, eNOS S1177D cDNA improved functional reserve of the LAD (33 +/- 5% vs. 7 +/- 3% of circumflex coronary artery-perfused myocardium), except for L-NAME coapplication (13 +/- 6%). CONCLUSIONS: Retroinfusion of eNOS S1177D cDNA induces neovascularization via endothelial cell proliferation and collateral growth. The resulting gain of perfusion enables an improved functional reserve of the hibernating myocardium.

Podocyte-specific deletion of integrin-linked kinase results in severe glomerular basement membrane alterations and progressive glomerulosclerosis.

Alterations in glomerular podocyte cell-cell and cell-matrix contacts are key events in progressive glomerular failure. Integrin-linked kinase (ILK) has been implicated in podocyte cell-matrix interaction and is induced in proteinuria. For evaluation of ILK function in vivo, mice with a Cre-mediated podocyte-specific ILK inactivation were generated. These mice seemed normal at birth but developed progressive focal segmental glomerulosclerosis and died in terminal renal failure. The first ultrastructural lesions that are seen at onset of albuminuria are glomerular basement membrane (GBM) alterations with a significant increase in true harmonic mean GBM thickness. Podocyte foot process effacement and loss of slit diaphragm followed with progression to unselective proteinuria. No significant reduction of slit membrane molecules (podocin and nephrin), key GBM components (fibronectin, laminins, and collagen IV isoforms), or podocyte integrins could be observed at onset of proteinuria. However, alpha3-integrins were relocalized into a granular pattern along the GBM, consistent with altered integrin-mediated matrix assembly in ILK-deficient podocytes. As the increased GBM thickness precedes structural podocyte lesions and key components of the GBM were expressed at comparable levels to controls, these data suggest an essential role of ILK for the close interconnection of GBM structure and podocyte function.

Does conventional cytogenetics detect the real frequency of 19q13 aberrations in benign thyroid lesions? A survey of 38 cases.

Structural aberrations involving chromosomal band 19q13.4 are examples of clonal cytogenetic deviations that have been detected in subgroups of follicular thyroid adenomas and goiters. About 45% of the adenomas and 8% of the goiters showed clonal aberrations, about 20% of which involve 19q13. The aberrations are translocations with a remarkable variation of the translocation partners of chromosome 19. Considering that structural changes involving small chromosome segments do not always have a characteristic band to be accurately identified, one may assume an even higher rate of these aberrations in thyroid lesions. To detect hidden 19q13 translocations, we performed fluorescence in situ hybridization analyses with a subtelomeric 19q specific probe on a subset of 38 thyroid adenomas with an apparently normal karyotype. No hidden chromosome abnormality was detected in our study, indicating that conventional cytogenetics reflects the true percentage of 19q13 aberrations in follicular thyroid adenomas.