- Email: [email protected]
342. Downregulation of Cardiomyocyte-Enriched Micrornas Contributes to Altered Gene Expression in Heart Failure
Cardiovascular Gene Therapy 340. AAV-Mediated Expression of VEGF165 and VEGF-B Enhances Cardiomyocytes Protection and Improves Heart Performance in the Infarcted Myocardium
Lorena Zentilin,1 Uday Puligadda,1 Serena Zacchigna,1 Lucia Pattarini,1 Vincenzo Lionetti,2 Fabio Recchia,2 Mauro Giacca.1 1 International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy; 2Scuola Superiore Sant’Anna, Sector of Medicine, Pisa, Italy.
A large body of evidence indicates that VEGF is an hypoxia-induced angiogenic factor indispensable for maintaining oxygen homeostasis in the heart by matching vascular density to cardiomyocyte metabolic demand. Indeed, we recently observed that the delivery of the VEGF165 gene using an AAV vector markedly improved cardiac function in an acute myocardial infarction model in chronically instrumented dogs (Ferrarini et al. 2006. Circ. Res. 98, 954). Most notably, however, this VEGF-based gene therapy approach determined a striking improvement in contractile function of the infarcted region as early as 48 hrs after gene delivery, suggesting that the beneficial effects of VEGF extended beyond its well-known angiogenic properties and possibly involved a direct effect on cardiomyocytes. Consistent with this observation, we found that isolated cardiomyocytes express abundant levels of the different VEGF receptors (VEGFR-1, VEGFR-2 and neuropilin-1), underscoring possible regulatory functions for the VEGF family members on these cells. To explore the functional role of these receptors, we investigated the effect of AAV vectors expressing VEGF165 (which bind to all receptors) and VEGF-B (a selective VEGFR-1 ligand) in a rat model of acute myocardial infarction. We found that both these vectors significantly improved cardiac performance compared to control animals. After 6 weeks from coronary artery ligation, continuous VEGF165 or VEGF-B expression determined a significant increase in LV anterior wall thickness, as determined by echocardiographic evaluation, in the absence of hypertrophy of the posterior wall. A thorough analysis revealed that VEGF had no effects on cardiomyocyte replication or regeneration of cardiac tissue after ischemic damage neither in vitro nor in vivo. Instead, both VEGF165 and VEGF-B significantly protected neonatal and adult rat cardiomyocytes from hypoxia-induced apoptosis and from death caused by the cardiotoxic drug epirubicin. Moreover, both VEGF165 and VEGF-B elicited a compensatory, cardioprotective hypertrophic transcriptional program in isolated cardiomyocytes as well as in heart tissue in vivo. Notably, both VEG165 and VEGF-B signalling through VEGFR-1 enhanced the expression of genes involved in the regulation of intracellular calcium transients such as RYR and SERCA2A, as well as of PGC-1a, a powerful regulator of mitochondrial metabolism and cardiac energetics. Taken together, these results indicate that the role VEGF in the heart extends beyond its angiogenic properties, and point to VEGFR-1 signalling as an essential component to induce cardiomyocyte protection both in vitro and in vivo.
341. Long Term Gene Expression Using AAV Vectors Unravels a Dual Role of Neuropilin1-Expressing Mononuclear Cells (NEMs) in Therapeutic and Pathological Angiogenesis
Serena Zacchigna,1 Alessandro Carrer,1 Lucia Pattarini,1 Lorena Zentilin,1 Milena Sinigaglia,1 Moimas Silvia,1 Gianfranco Sinagra,2 Mauro Giacca.1 1 International Centre for Genetic Engineering and Biotechnology, ICGEB, Trieste, Italy; 2Unità Operativa di Cardiologia, Azienda Ospedaliera-Universitaria “Ospedali Riuniti di Trieste, Trieste, Italy. Compelling evidence points toward a relevant role of bone marrow-derived mononuclear cells (BMCs) in blood vessel formation S128
and maturation. However, the available information is scanty and often controversial. On one side, the potential of BMCs to induce re-vascularization of ischemic tissues, either by recapitulating vasculogenesis in the adult or by remodelling small capillaries into large collateral arteries, has aroused much excitement. On the other side, BMCs almost invariably infiltrate any tumor mass and have been specifically associated to the vascular ‘normalization’ after administration of anti-angiogenic drugs, suggesting their involvement in vessel regression or maturation. To unravel the specific role of BMCs during the different steps of blood vessel biology, here we exploit the properties of AAV vectors to express for prolonged periods of time various cytokines differing in their property of attracting BMCs. In particular, we found that the intra-muscular injection of AAV vectors expressing either Sema3A or the 165 aa isoform of VEGF, determines a massive infiltration of the transduced tissues by a subset of BMCs expressing the neuropilin-1 (NP-1) receptor, which is shared by both ligands. These NP-1 expressing mononuclear cells (NEMs) were never incorporated into the vasculature formed in response to VEGF165, but their presence at the site of VEGF-induced angiogenesis was invariably paralleled by pericyte recruitment and formation of mature arteries. Consistently, clodronate-mediated depletion of mononuclear cells resulted in a lower number of arterial vessels at the site of VEGF overexpression. When recruited by Sema3A, NEMs were not angiogenic at all, and even inhibited ongoing angiogenesis, possibly by promoting endothelial cell apoptosis. In a xenogenic tumor model, the local overexpression of both VEGF165 and Sema3A exerted a potent inhibition on tumor growth. As these factors have distinct and opposite effects on endothelial cell proliferation and angiogenesis, it is likely that the observed antitumoral effect was essentially due to the infiltrating NEMs, equally recruited by both factors. At least three findings definitely support a relevant role of NP-1 in the recruitment of NEMs: i) the shorter VEGF121 isoform, which does not bind NP-1, is neither able to recruit NEMs nor to form mature arteries; ii) both VEGF165 and Sema3A act as chemoattractants for NEMs in vitro; iii) siRNA-mediated silencing of NP-1 significantly reduces NEM recruitment at the site of VEGF165-induced angiogenesis. Together, these findings sustain a model in which NEMs simultaneously inhibit endothelial cell proliferation and promote arterial vessel maturation, thus representing an attractive target for both therapeutic and tumorassociated angiogenesis.
342. Downregulation of CardiomyocyteEnriched Micrornas Contributes to Altered Gene Expression in Heart Failure
Sadakatsu Ikeda,1 Sek Won Kong,1 Jun Lu,2 Egbert Bisping,3 Natalya Bodyak,4 Kyu-Ho Lee,1,7 Qing Ma,1 Zinmar Aung,1 Peter M. Kang,4 Paul D. Allen,5 Todd Golub,2,6 William T. Pu.1 1 Children’s Hospital Boston and Harvard Medical School, Boston, MA; 2Dana Farber Cancer Institute and Broad Institute of Harvard and MIT, Boston, MA; 3Georg August University, Gottingen, Germany; 4Beth Israel Deaconess Medical Center and Harvard Medical School, Boston; 5Brigham and Women’s Hospital and Harvard Medical School, Boston; 6Howard Hughes Medical Institute, Chevy Chase; 7Medical University of South Carolina, Charleston, SC. Background: MicroRNAs (miRNAs) are a novel class of noncoding RNAs that regulate gene expression posttransciptionally. Altered miRNA expression has been implicated in diverse human diseases such as cancer. Accumulating evidence suggests the importance of miRNAs in the heart. However, the contribution of miRNAs to heart disease remains incompletely understood. Methods and Results: We measured the expression of 261 miRNAs in heart failure resulting from transgenic overexpression of calcineurin. 59 miRNAs were confidently detected in the heart, and 11 miRNAs belonging to 6 families (miR-1, -15, -30, -133, -195, -208) were Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
New Developments in Bone, Joint and Muscle Disease Gene/Cell Therapy downregulated compared to non-transgenic control (Welch’s t-test nominal p<0.05, false discovery rate <0.001). The results were validated by qRTPCR. There was no upregulated miRNA. Four of these miRNAs (miR-1, -30, -133, -208) were enriched in a purified cardiomyocyte preparation, compared to non-myocytes. Downregulation of these four miRNAs was reproduced in purified failing versus non-failing cardiomyocytes. This excluded artifactual downregulation from reduced myocyte fraction in failing hearts. The remaining two miRNAs (miR-15, and -195) were exclusively expressed in non-cardiomyocytes and did not changed in failing cardiomyocytes. Next, we used Affymetrix expression profiling to show that the predicted targets of these downregulated miRNAs were disproportionately upregulated compared to the entire transcriptome (Fisher’s exact p < 0.001). This suggests an association between downregulation of these miRNAs and upregulation of predicted target genes in heart failure. One particularly intriguing target of the predominant cardiac microRNA miR-1 is calmodulin, a key regulator of calcium signaling. We showed that calmodulin and downstream calmodulin signaling to NFAT is regulated by miR-1 in cultured cardiomyocytes. Furthermore, adenoviral delivery of miR-1 in vivo blocked cardiac hypertrophy in mouse hypertrophy model. Conclusion: Our results indicate that altered expression of cardiomyocyte-enriched miRNAs may contribute to abnormal gene expression in heart failure. The regulation of calmodulin and calcium signaling by miR-1 suggests a mechanism by which miR-1 may regulate heart function.
343. Endothelial Cell-Specific Rescue of Tie2 Angiopoietin Receptor in Tie2-Null Mice by microRNA-Regulated Lentiviral Vector Suggests a Requisite Role for Tie2 in the Hematopoietic System
Mary Anna Venneri,1 Daniela Biziato,1 Bernhard Gentner,1 Michele De Palma,1 Luigi Naldini.1,2 1 Angiogenesis and Tumor Targeting Unit and Telethon Istitute for Gene Therapy, San Raffaele Istitute, Milan, Italy; 2Vita-Salute San Raffaele University, Milan, Italy. The angiopoietin receptor Tie2 has important functions in the development and maintenance of the vascular system. In the adult, Tie2 is mainly expressed by endothelial cells (ECs), but also by distinct subsets of hematopoietic cells, including hematopoietic stem cells (HSCs). In this regard, we identified in mice and humans a population of Tie2-expressing monocytes (TEMs), which are proangiogenic and required for tumor vascularization in several models (De Palma et al., Cancer Cell, 2005; Venneri et al., Blood, 2007). Here, we report that TEMs appear early during embyogenesis, suggesting that they may have a role in organogenesis. Interestingly, TEMs migrate toward angiopoietin-2, suggesting that the angiopoietin/ Tie2 axis may regulate TEM activity. However, little is known about Tie2 function in the hematopoietic system. In fact, Tie2-null (Tie2-/-) mice exhibit severe vascular defects and die at E9.5, thus preventing the analysis of the role of Tie2 receptor in definitive hematopoiesis. To elucidate the role of Tie2 in hematopoietic cells, we have designed a new mouse model that lacks Tie2 specifically in the hematopoietic system (Tie2-/-hem). This model exploits the cell type-specific expression of endogenous microRNA to regulate transgene expression (Brown et al., Nat Med 2006). We inserted a sequence complementary to the hematopoietic-specific microRNA, miR-142-3p, into a lentiviral vector (LV) expressing the Tie2 receptor from a Tie2 promoter/enhancer (Tie2-Tie2-miR-142). The Tie2-Tie2miR-142 LV efficiently expressed functional Tie2 in ECs, but not in hematopoietic-lineage cells, while a control LV lacking the miR-1423p sequence (Tie2-Tie2) was expressed in both ECs and monocytic cells. We then transduced fertilized oocytes obtained by mating Tie2+/- heterozygous mice, either with the Tie2-Tie2-miR-142 LV, to Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
obtain Tie2-/-hem mice (hematopoietic knock-out; endothelial rescue), or with the Tie2-Tie2 LV, to obtain Tie2-/-rescue mice (hematopoietic and endothelial rescue). Whereas we successfully established several Tie2+/- mouse lines expressing the Tie2-Tie2-miR-142 transgene in ECs, we have not obtained to date any live Tie2-/-hem offspring, even by crossing Tie2+/- heterozygous mice carrying integrated Tie2-Tie2-miR-142 LV copies. Of note, FACS analyses and confocal microscopy performed on E7.5-E15.5 Tie2-GFP embryos showed that the reporter gene was expressed in both ECs and Tie2+F4/80+ TEMs, indicating effective vector expression in these cell types. In addition, a miR-142-regulated vector faithfully suppressed GFP expression in hematopoietic lineages, including HSCs, as shown by bone marrow transplantation experiments. Together, our findings suggest that Tie2 is required for the development and/or function of hematopoietic cells early during ontogenesis. We are currently investigating whether this requirement occurs in the HSC compartment, or for the development and activity of TEMs, or both.
New Developments in Bone, Joint and Muscle Disease Gene/Cell Therapy 344. Alpha 1 Antitrypsin (AAT) Gene Therapy in Collagen-Induced Arthritis
Christian Grimstein,1 Young-Kook Choi,1 Clive Wasserfall,2 Minoru Satoh,3 Mark Atkinson,2 Martha Campbell-Thompson,2 Sihong Song.1 1 Department of Pharmaceutics, University of Florida, Gainesville, FL; 2Department of Pathology, University of Florida, Gainesville, FL; 3Department of Medicine, University of Florida, Gainesville, FL. Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting ~1% of population in the United States. Although new biologicals that target TNF-alpha have dramatic effects in controlling disease activity in many patients, development of more efficient and safer treatment is necessary. Alpha-1 antitrypsin (AAT) is a multifunctional protein that has anti-inflammatory and tissue protective properties. In the present study we investigated the feasibility of AAT therapy for the treatment of RA in collagen-induced arthritis (CIA), a mouse model of RA. DBA/1 mice were immunized with bovine type II collagen on day 0 and boosted on day 21 to induce arthritis. These mice were intraperitoneally injected either with human (h)AAT (0.5mg/ injection, twice/week, starting from 1 week before immunization) or with recombinant adeno-associated virus vector expressing hAAT (rAAV8-CB-hAAT, 4x1011particles/mouse, injected 2 weeks before immunization). In addition, we also performed a combination therapy using hAAT gene therapy and doxycycline, a collagenase inhibitor (rAAV8-tet-on-hAAT, 2x1011particles/mouse, 200mg doxycycline/ kg body weight in food). Control groups received saline. Arthritis development was evaluated by incidence of arthritis, arthritic index (severity score 0~4), paw thickness, and number of arthritic paws. AAT protein therapy resulted in a hAAT serum level of 0.19 mg/ml and a significant suppression of arthritis development. At 44 days after immunization, incidence of severe arthritis in protein injected group (n=7) was 30% compared to 100% in control group (n=9). The average arthritic score in AAT treated group was significantly lower than that in control group (4.00±4.55 vs.8.29±3.20,p=0.044). In addition, ConA stimulated proliferation of splenocytes from AAT treated mice was significantly lower than that in control group (day 68,p<0.05). Similarly, rAAV8-mediated hAAT gene delivery resulted in a hAAT serum level of 0.1 mg/ml and significantly inhibited arthritis development. At 30 days after immunization, incidence of severe arthritis in vector injected group was 10% compared to 60% in control group (n=10). Average arthritic score in gene therapy group was significantly lower than that in control group (0.2±0.63 vs.2.7±1.29,p=0.007). rAAV8-hAAT gene therapy also resulted S129
Lorena Zentilin,1 Uday Puligadda,1 Serena Zacchigna,1 Lucia Pattarini,1 Vincenzo Lionetti,2 Fabio Recchia,2 Mauro Giacca.1 1 International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy; 2Scuola Superiore Sant’Anna, Sector of Medicine, Pisa, Italy.
A large body of evidence indicates that VEGF is an hypoxia-induced angiogenic factor indispensable for maintaining oxygen homeostasis in the heart by matching vascular density to cardiomyocyte metabolic demand. Indeed, we recently observed that the delivery of the VEGF165 gene using an AAV vector markedly improved cardiac function in an acute myocardial infarction model in chronically instrumented dogs (Ferrarini et al. 2006. Circ. Res. 98, 954). Most notably, however, this VEGF-based gene therapy approach determined a striking improvement in contractile function of the infarcted region as early as 48 hrs after gene delivery, suggesting that the beneficial effects of VEGF extended beyond its well-known angiogenic properties and possibly involved a direct effect on cardiomyocytes. Consistent with this observation, we found that isolated cardiomyocytes express abundant levels of the different VEGF receptors (VEGFR-1, VEGFR-2 and neuropilin-1), underscoring possible regulatory functions for the VEGF family members on these cells. To explore the functional role of these receptors, we investigated the effect of AAV vectors expressing VEGF165 (which bind to all receptors) and VEGF-B (a selective VEGFR-1 ligand) in a rat model of acute myocardial infarction. We found that both these vectors significantly improved cardiac performance compared to control animals. After 6 weeks from coronary artery ligation, continuous VEGF165 or VEGF-B expression determined a significant increase in LV anterior wall thickness, as determined by echocardiographic evaluation, in the absence of hypertrophy of the posterior wall. A thorough analysis revealed that VEGF had no effects on cardiomyocyte replication or regeneration of cardiac tissue after ischemic damage neither in vitro nor in vivo. Instead, both VEGF165 and VEGF-B significantly protected neonatal and adult rat cardiomyocytes from hypoxia-induced apoptosis and from death caused by the cardiotoxic drug epirubicin. Moreover, both VEGF165 and VEGF-B elicited a compensatory, cardioprotective hypertrophic transcriptional program in isolated cardiomyocytes as well as in heart tissue in vivo. Notably, both VEG165 and VEGF-B signalling through VEGFR-1 enhanced the expression of genes involved in the regulation of intracellular calcium transients such as RYR and SERCA2A, as well as of PGC-1a, a powerful regulator of mitochondrial metabolism and cardiac energetics. Taken together, these results indicate that the role VEGF in the heart extends beyond its angiogenic properties, and point to VEGFR-1 signalling as an essential component to induce cardiomyocyte protection both in vitro and in vivo.
341. Long Term Gene Expression Using AAV Vectors Unravels a Dual Role of Neuropilin1-Expressing Mononuclear Cells (NEMs) in Therapeutic and Pathological Angiogenesis
Serena Zacchigna,1 Alessandro Carrer,1 Lucia Pattarini,1 Lorena Zentilin,1 Milena Sinigaglia,1 Moimas Silvia,1 Gianfranco Sinagra,2 Mauro Giacca.1 1 International Centre for Genetic Engineering and Biotechnology, ICGEB, Trieste, Italy; 2Unità Operativa di Cardiologia, Azienda Ospedaliera-Universitaria “Ospedali Riuniti di Trieste, Trieste, Italy. Compelling evidence points toward a relevant role of bone marrow-derived mononuclear cells (BMCs) in blood vessel formation S128
and maturation. However, the available information is scanty and often controversial. On one side, the potential of BMCs to induce re-vascularization of ischemic tissues, either by recapitulating vasculogenesis in the adult or by remodelling small capillaries into large collateral arteries, has aroused much excitement. On the other side, BMCs almost invariably infiltrate any tumor mass and have been specifically associated to the vascular ‘normalization’ after administration of anti-angiogenic drugs, suggesting their involvement in vessel regression or maturation. To unravel the specific role of BMCs during the different steps of blood vessel biology, here we exploit the properties of AAV vectors to express for prolonged periods of time various cytokines differing in their property of attracting BMCs. In particular, we found that the intra-muscular injection of AAV vectors expressing either Sema3A or the 165 aa isoform of VEGF, determines a massive infiltration of the transduced tissues by a subset of BMCs expressing the neuropilin-1 (NP-1) receptor, which is shared by both ligands. These NP-1 expressing mononuclear cells (NEMs) were never incorporated into the vasculature formed in response to VEGF165, but their presence at the site of VEGF-induced angiogenesis was invariably paralleled by pericyte recruitment and formation of mature arteries. Consistently, clodronate-mediated depletion of mononuclear cells resulted in a lower number of arterial vessels at the site of VEGF overexpression. When recruited by Sema3A, NEMs were not angiogenic at all, and even inhibited ongoing angiogenesis, possibly by promoting endothelial cell apoptosis. In a xenogenic tumor model, the local overexpression of both VEGF165 and Sema3A exerted a potent inhibition on tumor growth. As these factors have distinct and opposite effects on endothelial cell proliferation and angiogenesis, it is likely that the observed antitumoral effect was essentially due to the infiltrating NEMs, equally recruited by both factors. At least three findings definitely support a relevant role of NP-1 in the recruitment of NEMs: i) the shorter VEGF121 isoform, which does not bind NP-1, is neither able to recruit NEMs nor to form mature arteries; ii) both VEGF165 and Sema3A act as chemoattractants for NEMs in vitro; iii) siRNA-mediated silencing of NP-1 significantly reduces NEM recruitment at the site of VEGF165-induced angiogenesis. Together, these findings sustain a model in which NEMs simultaneously inhibit endothelial cell proliferation and promote arterial vessel maturation, thus representing an attractive target for both therapeutic and tumorassociated angiogenesis.
342. Downregulation of CardiomyocyteEnriched Micrornas Contributes to Altered Gene Expression in Heart Failure
Sadakatsu Ikeda,1 Sek Won Kong,1 Jun Lu,2 Egbert Bisping,3 Natalya Bodyak,4 Kyu-Ho Lee,1,7 Qing Ma,1 Zinmar Aung,1 Peter M. Kang,4 Paul D. Allen,5 Todd Golub,2,6 William T. Pu.1 1 Children’s Hospital Boston and Harvard Medical School, Boston, MA; 2Dana Farber Cancer Institute and Broad Institute of Harvard and MIT, Boston, MA; 3Georg August University, Gottingen, Germany; 4Beth Israel Deaconess Medical Center and Harvard Medical School, Boston; 5Brigham and Women’s Hospital and Harvard Medical School, Boston; 6Howard Hughes Medical Institute, Chevy Chase; 7Medical University of South Carolina, Charleston, SC. Background: MicroRNAs (miRNAs) are a novel class of noncoding RNAs that regulate gene expression posttransciptionally. Altered miRNA expression has been implicated in diverse human diseases such as cancer. Accumulating evidence suggests the importance of miRNAs in the heart. However, the contribution of miRNAs to heart disease remains incompletely understood. Methods and Results: We measured the expression of 261 miRNAs in heart failure resulting from transgenic overexpression of calcineurin. 59 miRNAs were confidently detected in the heart, and 11 miRNAs belonging to 6 families (miR-1, -15, -30, -133, -195, -208) were Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
New Developments in Bone, Joint and Muscle Disease Gene/Cell Therapy downregulated compared to non-transgenic control (Welch’s t-test nominal p<0.05, false discovery rate <0.001). The results were validated by qRTPCR. There was no upregulated miRNA. Four of these miRNAs (miR-1, -30, -133, -208) were enriched in a purified cardiomyocyte preparation, compared to non-myocytes. Downregulation of these four miRNAs was reproduced in purified failing versus non-failing cardiomyocytes. This excluded artifactual downregulation from reduced myocyte fraction in failing hearts. The remaining two miRNAs (miR-15, and -195) were exclusively expressed in non-cardiomyocytes and did not changed in failing cardiomyocytes. Next, we used Affymetrix expression profiling to show that the predicted targets of these downregulated miRNAs were disproportionately upregulated compared to the entire transcriptome (Fisher’s exact p < 0.001). This suggests an association between downregulation of these miRNAs and upregulation of predicted target genes in heart failure. One particularly intriguing target of the predominant cardiac microRNA miR-1 is calmodulin, a key regulator of calcium signaling. We showed that calmodulin and downstream calmodulin signaling to NFAT is regulated by miR-1 in cultured cardiomyocytes. Furthermore, adenoviral delivery of miR-1 in vivo blocked cardiac hypertrophy in mouse hypertrophy model. Conclusion: Our results indicate that altered expression of cardiomyocyte-enriched miRNAs may contribute to abnormal gene expression in heart failure. The regulation of calmodulin and calcium signaling by miR-1 suggests a mechanism by which miR-1 may regulate heart function.
343. Endothelial Cell-Specific Rescue of Tie2 Angiopoietin Receptor in Tie2-Null Mice by microRNA-Regulated Lentiviral Vector Suggests a Requisite Role for Tie2 in the Hematopoietic System
Mary Anna Venneri,1 Daniela Biziato,1 Bernhard Gentner,1 Michele De Palma,1 Luigi Naldini.1,2 1 Angiogenesis and Tumor Targeting Unit and Telethon Istitute for Gene Therapy, San Raffaele Istitute, Milan, Italy; 2Vita-Salute San Raffaele University, Milan, Italy. The angiopoietin receptor Tie2 has important functions in the development and maintenance of the vascular system. In the adult, Tie2 is mainly expressed by endothelial cells (ECs), but also by distinct subsets of hematopoietic cells, including hematopoietic stem cells (HSCs). In this regard, we identified in mice and humans a population of Tie2-expressing monocytes (TEMs), which are proangiogenic and required for tumor vascularization in several models (De Palma et al., Cancer Cell, 2005; Venneri et al., Blood, 2007). Here, we report that TEMs appear early during embyogenesis, suggesting that they may have a role in organogenesis. Interestingly, TEMs migrate toward angiopoietin-2, suggesting that the angiopoietin/ Tie2 axis may regulate TEM activity. However, little is known about Tie2 function in the hematopoietic system. In fact, Tie2-null (Tie2-/-) mice exhibit severe vascular defects and die at E9.5, thus preventing the analysis of the role of Tie2 receptor in definitive hematopoiesis. To elucidate the role of Tie2 in hematopoietic cells, we have designed a new mouse model that lacks Tie2 specifically in the hematopoietic system (Tie2-/-hem). This model exploits the cell type-specific expression of endogenous microRNA to regulate transgene expression (Brown et al., Nat Med 2006). We inserted a sequence complementary to the hematopoietic-specific microRNA, miR-142-3p, into a lentiviral vector (LV) expressing the Tie2 receptor from a Tie2 promoter/enhancer (Tie2-Tie2-miR-142). The Tie2-Tie2miR-142 LV efficiently expressed functional Tie2 in ECs, but not in hematopoietic-lineage cells, while a control LV lacking the miR-1423p sequence (Tie2-Tie2) was expressed in both ECs and monocytic cells. We then transduced fertilized oocytes obtained by mating Tie2+/- heterozygous mice, either with the Tie2-Tie2-miR-142 LV, to Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
obtain Tie2-/-hem mice (hematopoietic knock-out; endothelial rescue), or with the Tie2-Tie2 LV, to obtain Tie2-/-rescue mice (hematopoietic and endothelial rescue). Whereas we successfully established several Tie2+/- mouse lines expressing the Tie2-Tie2-miR-142 transgene in ECs, we have not obtained to date any live Tie2-/-hem offspring, even by crossing Tie2+/- heterozygous mice carrying integrated Tie2-Tie2-miR-142 LV copies. Of note, FACS analyses and confocal microscopy performed on E7.5-E15.5 Tie2-GFP embryos showed that the reporter gene was expressed in both ECs and Tie2+F4/80+ TEMs, indicating effective vector expression in these cell types. In addition, a miR-142-regulated vector faithfully suppressed GFP expression in hematopoietic lineages, including HSCs, as shown by bone marrow transplantation experiments. Together, our findings suggest that Tie2 is required for the development and/or function of hematopoietic cells early during ontogenesis. We are currently investigating whether this requirement occurs in the HSC compartment, or for the development and activity of TEMs, or both.
New Developments in Bone, Joint and Muscle Disease Gene/Cell Therapy 344. Alpha 1 Antitrypsin (AAT) Gene Therapy in Collagen-Induced Arthritis
Christian Grimstein,1 Young-Kook Choi,1 Clive Wasserfall,2 Minoru Satoh,3 Mark Atkinson,2 Martha Campbell-Thompson,2 Sihong Song.1 1 Department of Pharmaceutics, University of Florida, Gainesville, FL; 2Department of Pathology, University of Florida, Gainesville, FL; 3Department of Medicine, University of Florida, Gainesville, FL. Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting ~1% of population in the United States. Although new biologicals that target TNF-alpha have dramatic effects in controlling disease activity in many patients, development of more efficient and safer treatment is necessary. Alpha-1 antitrypsin (AAT) is a multifunctional protein that has anti-inflammatory and tissue protective properties. In the present study we investigated the feasibility of AAT therapy for the treatment of RA in collagen-induced arthritis (CIA), a mouse model of RA. DBA/1 mice were immunized with bovine type II collagen on day 0 and boosted on day 21 to induce arthritis. These mice were intraperitoneally injected either with human (h)AAT (0.5mg/ injection, twice/week, starting from 1 week before immunization) or with recombinant adeno-associated virus vector expressing hAAT (rAAV8-CB-hAAT, 4x1011particles/mouse, injected 2 weeks before immunization). In addition, we also performed a combination therapy using hAAT gene therapy and doxycycline, a collagenase inhibitor (rAAV8-tet-on-hAAT, 2x1011particles/mouse, 200mg doxycycline/ kg body weight in food). Control groups received saline. Arthritis development was evaluated by incidence of arthritis, arthritic index (severity score 0~4), paw thickness, and number of arthritic paws. AAT protein therapy resulted in a hAAT serum level of 0.19 mg/ml and a significant suppression of arthritis development. At 44 days after immunization, incidence of severe arthritis in protein injected group (n=7) was 30% compared to 100% in control group (n=9). The average arthritic score in AAT treated group was significantly lower than that in control group (4.00±4.55 vs.8.29±3.20,p=0.044). In addition, ConA stimulated proliferation of splenocytes from AAT treated mice was significantly lower than that in control group (day 68,p<0.05). Similarly, rAAV8-mediated hAAT gene delivery resulted in a hAAT serum level of 0.1 mg/ml and significantly inhibited arthritis development. At 30 days after immunization, incidence of severe arthritis in vector injected group was 10% compared to 60% in control group (n=10). Average arthritic score in gene therapy group was significantly lower than that in control group (0.2±0.63 vs.2.7±1.29,p=0.007). rAAV8-hAAT gene therapy also resulted S129