Interleukin-2 enhances angiogenesis and preserves cardiac function following myocardial infarction.

We previously demonstrated that injection of IL-2-activated natural killer (NK) cells contribute to vascular remodeling via a4b7 integrin and killer cell lectin-like receptor (KLRG) 1 and promote cardiac repair following myocardial infarction (MI). The aim of the present study is to test the hypothesis that injection of recombinant human interleukin (rhIL)-2 improves angiogenesis and preserves heart function after MI. A single IV injection of rhIL-2 two days following MI improved by 27.7% the left ventricular (LV) fractional shortening of immune competent (C57Bl6) mice, but had no effect on cardiac function of immune-deficient (NOD-SCID IL2Rγnull) mice. Immunohistochemical analysis of C57Bl6 cross sections of heart revealed that collagen deposition was reduced by 23.1% and that capillary density was enhanced in the scar area and the border zone of the infarct respectively by 22.4% and 33.6% following rhIL-2 injection. In addition, rhIL-2 enhanced 1.6-fold the in vivo endothelial cell proliferation index and 1.8-fold the number of NK cell infiltrating the infarcted heart, but had no effect on the number of cardiac CD4 and CD8 cells. In vitro, rhIL-2 activated NK cells enhanced cardiac endothelial cell proliferation by 17.2%. Here we show that a single IV injection of rhIL-2 positively impacted cardiac function by improving angiogenesis through a process involving NK cells.

Vascular endothelial growth factor reduced hypoxia-induced death of human myoblasts and improved their engraftment in mouse muscles.

Muscle precursor cell (myoblasts) transplantation is considered as a potential approach to restore dystrophin expression in Duchenne muscular dystrophy (DMD) patients. The study purpose was to verify the implication of hypoxia in the myoblast death observed after their transplantation and also to evaluate the potential beneficial effects of vascular endothelial growth factor (VEGF) overexpression on myoblast engraftment in a murine model. Pimonidazole hydrochloride (hypoxyprobe-1) was used to mark selectively myoblasts to evaluate their hypoxia in vivo. In vitro, hypoxia was induced by culturing human myoblasts in hypoxic environment. In vitro effects of VEGF(165) on survival of human cells was assessed by Hoescht-PI labeling. Tibialis anterior (TA) female mouse muscles were electroporated with a plasmid containing the VEGF(165) or with an empty vector. Circulating VEGF concentration was assessed by ELISA. After 2 weeks of electroporation, severe combined immunodeficient (SCID) mice were transplanted with 800 000 human male myoblasts labeled with radioactive thymidine. Mouse muscles were harvested 2 and 4 days later and myoblast survival and proliferation were evaluated by scintigraphy and Y chromosome quantitative PCR. The long-term graft success was evaluated using gamma-radiograph imaging and by counting the dystrophin positive muscle fibers. Hypoxyprobe labeling has shown that most of the transplanted myoblasts were hypoxic. The transplantation of radioactive male myoblasts in female mice electroporated with the VEGF(165) plasmid demonstrated that VEGF reduced their death by 10% but did not improve their proliferation. VEGF(165) enhanced human myoblast survival in vitro under hypoxic conditions. Electroporation of TA muscles of SCID mouse with the vector coding for VEGF(165) promoted angiogenesis and improved by 1.5-fold the success of myoblast transplantation in comparison with the control mice that were electroporated with the empty vector. These results indicate that hypoxia is partially responsible for the death of the transplanted myoblasts. VEGF can be used to improve myoblast survival and the graft success.

[Aneurysm of the coeliac trunk. A case report]. / Anévrysme du tronc coeliaque. A propos d'un cas.

Aneurysm of the coeliac artery is a rare vascular problem. The most serious clinical complication of coeliac artery aneurysm is rupture. Because of this, surgery is traditionally recommended. This paper presents a case of a coeliac artery aneurysm treated by open surgery.

A novel and simplified method of culture of human blood-derived early endothelial progenitor cells for the treatment of ischemic vascular disease.

Endothelial progenitor cells (EPCs) consist of two different subpopulations named early (eEPCs) and late EPCs (lEPCs) that are derived from CD14(+) and CD14(-) circulating cells, respectively. These cells are regularly cultured over fibronectin-coated surfaces in endothelial basal medium (EBM)-2 supplemented with insulin-like growth factor (IGF-1), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF). We have developed a new and simplified method for culturing human EPCs obtained from peripheral blood and tested their ability to preserve cardiac function following infarction. We first demonstrated that eEPCs derived from human peripheral blood mononuclear cells (PBMCs) and cultured in EBM-2 medium supplemented with autologous serum (10%) over fibronectin-coated surfaces (10 µg/ml) in the presence of IGF-1 (50 ng/ml) only, have a secretome similar to eEPCs cultured under regular conditions with IGF-1, VEGF, EGF, and FGF. Our data also indicate that IGF-1 modulates PBMC secretome in a dose-dependent manner. In another series of experiments, we showed that PBMCs cultured in suspension in bags (S-PBMCs) in basal medium supplemented with fibronectin and IGF-1 secrete significant amounts of stem cell factor (SCF, 31.3 ± 3.1 pg/ml)), hepatocyte growth factor (HGF, 438.6 ± 41.4 pg/ml), soluble tumor necrosis factor receptor 1 (sTNFR1, 127.1 ± 9.9 pg/ml), VEGF (139.3 ± 9.6 pg/ml), and IGF-1 (147.2 ± 46.1 pg/ml) but very low levels of TNF-α (13.4 ± 2.5 pg/ml). S-PBMCs injected intravenously into NOD SCID mice migrated to the injured myocardium, reduced cardiac fibrosis, enhanced angiogenesis, and preserved cardiac function after myocardial infarction (MI) in a manner similar to eEPCs cultured under standard conditions. In conclusion, we show in this study a refined and optimized method for culturing eEPCs. Our data indicate that S-PBMCs are composed of several cell populations including eEPCs and that they secrete high amounts of antiapoptotic, anti-inflammatory, and proangiogenic factors capable of preserving cardiac function following MI.

Monocyte derivatives promote angiogenesis and myocyte survival in a model of myocardial infarction.

In this study, we have investigated the hypothesis that previously reported beneficial effect of peripheral blood mononuclear cells cultured under angiogenic conditions on cardiovascular function following ischemia is not limited to EPCs but also to monocytes contained therein. We first purified and analyzed the phenotype and secretome of human and murine blood monocytes cultured under angiogenic conditions (named MDs for monocyte derivatives) and tested their effect in a mouse model of myocardial infarction (MI). FACS analysis of MDs shows that these cells express mature endothelial cell markers and that their proliferative capacity is virtually absent, consistent with their end-differentiated monocytic ontogeny. MDs secreted significant levels of HGF, IGF-1, MCP-1, and sTNFR-1 relative to their monocyte precursors. MDs were unable to form vascular networks in vitro when cultured on matrix coated flasks. Treatment of murine HL-1 cardiomyocyte cell line with MD-conditioned medium reduced their death induced by TNF-alpha, staurosporine, and oxidative stress, and this effect was dependent upon MD-derived sTNFR-1, HGF, and IGF-1. We further demonstrate that MD secretome promoted endothelial cell proliferation and capacity to form vessels in vitro and this was dependent upon MD-derived MCP-1, HGF, and IGF-1. Echocardiography analysis showed that MD myocardial implantation improved left ventricle fractional shortening of mouse hearts following MI and was associated with reduced myocardial fibrosis and enhancement of angiogenesis. Transplanted MDs and their secretome participate in preserving functional myocardium after ischemic insult and attenuate pathological remodeling.

Overexpression of follistatin in human myoblasts increases their proliferation and differentiation, and improves the graft success in SCID mice.

Duchenne muscular dystrophy is caused by the absence of functional dystrophin, leading to the myofiber membrane instability and progressive muscle atrophy. Myoblast transplantation in dystrophic muscles is a potential therapy, as it permits the long-term restoration of dystrophin expression in transplanted muscles. However, the success of this approach is limited by the short period of muscle repair following myoblast transplantation. Myostatin, a powerful inhibitor of muscle growth, is involved in terminating the period of muscle repair following injury by reducing myoblast proliferation and differentiation. Follistatin forms a complex with myostatin, preventing its interaction with its receptor and thus blocking the myostatin signal. Here, we used a lentivirus to overexpress the follistatin protein in normal myoblasts to block the myostatin signaling. We measured the potential of transduced myoblasts to proliferate and to form multinucleated myotubes in vitro. And finally, we considered the engraftment success of those transduced myoblasts in comparison with control cells in vivo within SCID mice TA muscle. Our results first confirmed the overexpression of follistatin into lentivirus transduced myoblasts, and second showed that the overexpression of the follistatin in normal human myoblasts improved in vitro their proliferation rate by about 1.5-fold after 96 h and also their differentiation rate by about 1.6- and 1.8-fold, respectively, in the absence and in the presence of recombinant myostatin. Finally, our data demonstrated that the engraftment of human normal myoblasts overexpressing the follistatin protein into SCID mouse muscles was enhanced by twofold.

Induction of Anoikis following myoblast transplantation into SCID mouse muscles requires the Bit1 and FADD pathways.

Seventy-five percent of the myoblasts transplanted in the mouse muscle die during the first 4 days following transplantation. The purpose of this study was to determine if anoikis plays a role in this phenomenon. Survival and proliferation of myoblasts in vitro were determined by Hoescht-PI labeling and cell counts respectively. In vivo cell survival and proliferation were quantified by injecting human male myoblasts labeled with (14)C-thymidine in SCID mouse muscles. Survival and proliferation of the transplanted myoblasts were evaluated by scintigraphy and quantitative PCR of human Y chromosomal DNA. Inclusion of the extracellular matrix protein fibronectin enhanced transplanted myoblast survival by 1.7-fold while vitronectin improved their proliferation by 1.8-fold. Reductions in FADD and Bit1 expression reduced anoikis in vitro and improved the injected myoblast survival in vivo. Ectopic expression of the anti-apoptotic protein Bcl-2 completely abolished myoblast anoikis in vitro and enhanced cell survival by 3.1-fold in vivo. Cell death following transplantation appears to me mediated in part by anoikis. Inclusion of extracellular matrix proteins enhanced both survival and proliferation. Reduced expression of the proapoptotic proteins Bit1 and FADD or overexpression of Bcl-2 improved myoblast survival.

A synthetic mechano growth factor E Peptide enhances myogenic precursor cell transplantation success.

Myogenic precursor cell (MPC) transplantation is a good strategy to introduce dystrophin expression in muscles of Duchenne muscular dystrophy (DMD) patients. Insulin-like growth factor (IGF-1) promotes MPC activities, such as survival, proliferation, migration and differentiation, which could enhance the success of their transplantation. Alternative splicing of the IGF-1 mRNA produces different muscle isoforms. The mechano growth factor (MGF) is an isoform, especially expressed after a mechanical stress. A 24 amino acids peptide corresponding to the C-terminal part of the MGF E domain (MGF-Ct24E peptide) was synthesized. This peptide had been shown to enhance the proliferation and delay the terminal differentiation of C(2)C(12) myoblasts. The present study showed that the MGF-Ct24E peptide improved human MPC transplantation by modulating their proliferation and differentiation. Indeed, intramuscular or systemic delivery of this synthetic peptide significantly promoted engraftment of human MPCs in mice. In vitro experiments demonstrated that the MGF-Ct24E peptide enhanced MPC proliferation by a different mechanism than the binding to the IGF-1 receptor. Moreover, MGF-Ct24E peptide delayed human MPC differentiation while having no outcome on survival. Those combined effects are probably responsible for the enhanced transplantation success. Thus, the MGF-Ct24E peptide is an interesting agent to increase MPC transplantation success in DMD patients.

Interleukin-4 improves the migration of human myogenic precursor cells in vitro and in vivo.

Different molecules are available to recruit new neighboring myogenic cells to the site of regeneration. Formerly called B cell stimulatory factor-1, IL-4 can now be included in the list of motogenic factors. The present report demonstrates that human IL-4 is not required for fusion between mononucleated myoblasts but is required for myotube maturation. In identifying IL-4 as a pro-migratory agent for myogenic cells, these results provide a mechanism which partly explains IL-4 demonstrated activity during differentiation. Among the different mechanisms by which IL-4 might enhance myoblast migration processes, our results indicate that there are implications of some integrins and of three major components of the fibrinolytic system. Indeed, increases in the amount of active urokinase plasminogen activator and its receptor were observed following an IL-4 treatment, while the plasminogen activator inhibitor-1 decreased. Finally, IL-4 did not modify the amount of cell surface alpha5 integrin but increased the presence of beta3 and beta1 integrins. This integrin modulation might favor myogenic cell migration and its interaction with newly formed myotubes. Therefore, IL-4 co-injection with transplanted myoblasts might be an approach to enhance the migration of transplanted cells for the treatment of a damaged myocardium or of a Duchenne Muscular Dystrophy patient.

Tubulyzine, a novel tri-substituted triazine, prevents the early cell death of transplanted myogenic cells and improves transplantation success.

Myoblast transplantation (MT) is a potential therapeutic approach for several muscular dystrophies. A major limiting factor is that only a low percentage of the transplanted myoblasts survives the procedure. Recent advances regarding how and when the myoblasts die indicate that events preceding actual tissue implantation and during the first days after the transplantation are crucial. Myoseverin, a recently identified tri-substituted purine, was shown to induce in vitro the fission of multinucleated myotubes and affect the expression of a variety of growth factors, and immunomodulation, extracellular matrix-remodeling, and stress response genes. Since the effects of myoseverin are consistent with the activation of pathways involved in wound healing and tissue regeneration, we have investigated whether pretreatment and co-injection of myoblasts with Tubulyzine (microtubule lysing triazine), an optimized myoseverin-like molecule recently identified from a triazine library, could reduce myoblast cell death following their transplantation and consequently improves the success of myoblast transplantation. In vitro, using annexin-V labeling, we showed that Tubulyzine (5 microM) prevents normal myoblasts from apoptosis induced by staurosporine (1 microM). In vivo, the pretreatment and co-injection of immortal and normal myoblasts with Tubulyzine reduced significantly cell death (assessed by the radio-labeled thymidine of donor DNA) and increased survival of myoblasts transplanted in Tibialis anterior (TA) muscles of mdx mice, thus giving rise to more hybrid myofibers compared to transplanted untreated cells. Our results suggest that Tubulyzine can be used as an in vivo survival factor to improve the myoblast-mediated gene transfer approach.