Annexin A2 is a molecular target for TM601, a peptide with tumor-targeting and anti-angiogenic effects.

TM601 is a synthetic form of chlorotoxin, a 36-amino acid peptide derived from the venom of the Israeli scorpion, Leirius quinquestriatus, initially found to specifically bind and inhibit the migration of glioma cells in culture. Subsequent studies demonstrated specific in vitro binding to additional tumor cell lines. Recently, we demonstrated that proliferating human vascular endothelial cells are the only normal cell line tested that exhibits specific binding to TM601. Here, we identify annexin A2 as a novel binding partner for TM601 in multiple human tumor cell lines and human umbilical vein endothelial cell (HUVEC). We demonstrate that the surface binding of TM601 to the pancreatic tumor cell line Panc-1 is dependent on the expression of annexin A2. Identification of annexin A2 as a binding partner for TM601 is also consistent with the anti-angiogenic effects of TM601. Annexin A2 functions in angiogenesis by binding to tissue plasminogen activator and regulating plasminogen activation on vascular endothelial cells. We demonstrate that in HUVECs, TM601 inhibits both vascular endothelial growth factor- and basic fibroblast growth factor-induced tissue plasminogen activator activation, which is required for activation of plasminogen to plasmin. Consistent with inhibition of cell surface protease activity, TM601 also inhibits platelet-derived growth factor-C induced trans-well migration of both HUVEC and U373-MG glioma cells.

Agents that bind annexin A2 suppress ocular neovascularization.

TM601 is a synthetic polypeptide with sequence derived from the venom of the scorpion Leiurus quinquestriatus that has anti-neoplastic activity. It has recently been demonstrated to bind annexin A2 on cultured tumor and vascular endothelial cells and to suppress blood vessel growth on chick chorioallantoic membrane. In this study, we investigated the effects of TM601 in models of ocular neovascularization (NV). When administered by intraocular injection, intravenous injections, or periocular injections, TM601 significantly suppressed the development of choroidal NV at rupture sites in Bruch's membrane. Treatment of established choroidal NV with TM601 caused apoptosis of endothelial cells and regression of the NV. TM601 suppressed ischemia-induced and vascular endothelial growth factor-induced retinal NV and reduced excess vascular permeability induced by vascular endothelial growth factor. Immunostaining with an antibody directed against TM601 showed that after intraocular or periocular injection, TM601 selectively bound to choroidal or retinal NV and co-localized with annexin A2, which is undetectable in normal retinal and choroidal vessels, but is upregulated in endothelial cells participating in choroidal or retinal NV. Intraocular injection of plasminogen or tissue plasminogen activator, which like TM601 bind to annexin A2, also suppressed retinal NV. This study supports the hypothesis that annexin A2 is an important target for treatment of neovascular diseases and suggests that TM601, through its interaction with annexin A2, causes suppression and regression of ocular NV and reduces vascular leakage and thus may provide a new treatment for blinding diseases such as neovascular age-related macular degeneration and diabetic retinopathy.

Targeted delivery of antitumoral therapy to glioma and other malignancies with synthetic chlorotoxin (TM-601).

Targeted therapies for cancer is a rapidly advancing field, but the identification of tumor-specific ligands has proven difficult. Chlorotoxin (CTX) is a small, 36 amino acid neurotoxin isolated from the venom of the Giant Yellow Israeli scorpion Leiurus Quinquestriatus. Interestingly, the peptide has been found to preferentially bind to a variety of human malignancies, but shows little or no binding to normal human tissues. A synthetic version of this peptide (TM-601) has been manufactured and covalently linked to iodine 131 (131I-TM-601) as a means of targeting radiation to tumor cells. Preclinical studies and Phase I clinical trials have been completed in patients with recurrent glioma, a type of malignant brain tumor. These studies demonstrated that intracavitary dosing of 131I-TM-601 appears safe, minimally toxic, and binds malignant glioma with high affinity and for long durations. A Phase II trial of this agent using higher doses of radioactivity and repeated local administrations is underway. In addition, enrolment has begun in a Phase I trial evaluating whether systemically delivered 131I-TM-601 can be used to image metastatic solid tumors and primary gliomas. Due to its small size, selective tumor binding properties, minimal toxicity and relative ease of manipulation, CTX represents a potentially important targeting agent for many cancers.

Safety and feasibility of percutaneous autologous skeletal myoblast transplantation in the coil-infarcted swine myocardium.

INTRODUCTION: Autologous skeletal myoblast transplantation (ASMT) for myocardial regeneration is a promising new treatment for patients with congestive heart failure secondary to myocardial infarction (MI). However, non-surgical delivery could broaden the utility of this approach. The present study was designed to evaluate the safety and feasibility of transplanting autologous skeletal myoblast (ASM) via endovascular delivery into the infarcted swine myocardium. METHODS: Seven female Yorkshire swine successfully underwent induced left ventricular MI. ASM biopsies were obtained from the hind limb of each animal and myoblasts were expanded in vitro. In a pilot experiment, ASM were labeled with iridium and short-term retention and biodistribution was determined 2 h after ASM delivery via the MyoStar needle-injection catheter inserted through the femoral artery. At 30 days post-infarction, the remaining animals were divided into three groups containing 2 animals each for percutaneous catheter delivery into the infarcted zone: group 1 control animals were injected with media only, group 2 and 3 animals were injected with approximately 300 x 10(6) and 600 x 10(6) ASM, respectively. Sixty days post-transplantation, the swine hearts were harvested. RESULTS: During the 60-day period between transplantation and harvest, no adverse events were recorded, and continuous rhythm monitoring revealed no arrhythmias. In the small sampling size, myocardial function assessments revealed a trend toward improvement in the treatment groups with respect to ejection fraction, viability, and cardiac index. However, histology of treated swine hearts identified no skeletal muscle cells. DISCUSSION: Percutaneous ASMT into an infarcted swine myocardium is feasible and safe, and may contribute to overall improved heart function.

Autologous skeletal myoblasts transplanted to ischemia-damaged myocardium in humans. Histological analysis of cell survival and differentiation.

OBJECTIVES: We report histological analysis of hearts from patients with end-stage heart disease who were transplanted with autologous skeletal myoblasts concurrent with left ventricular assist device (LVAD) implantation. BACKGROUND: Autologous skeletal myoblast transplantation is under investigation as a means to repair infarcted myocardium. To date, there is only indirect evidence to suggest survival of skeletal muscle in humans. METHODS: Five patients (all male; median age 60 years) with ischemic cardiomyopathy, refractory heart failure, and listed for heart transplantation underwent muscle biopsy from the quadriceps muscle. The muscle specimen was shipped to a cell isolation facility where myoblasts were isolated and grown. Patients received a transplant of 300 million cells concomitant with LVAD implantation. Four patients underwent LVAD explant after 68, 91, 141, and 191 days of LVAD support (three transplant, one LVAD death), respectively. One patient remains alive on LVAD support awaiting heart transplantation. RESULTS: Skeletal muscle cell survival and differentiation into mature myofibers were directly demonstrated in scarred myocardium from three of the four explanted hearts using an antibody against skeletal muscle-specific myosin heavy chain. An increase in small vessel formation was observed in one of three patients at the site of surviving myotubes, but not in adjacent tissue devoid of engrafted cells. CONCLUSIONS: These findings represent demonstration of autologous myoblast cell survival in human heart. The implanted skeletal myoblasts formed viable grafts in heavily scarred human myocardial tissue. These results establish the feasibility of myoblast transplants for myocardial repair in humans.

Triple immune suppression increases short-term survival of porcine fetal retinal pigment epithelium xenografts.

PURPOSE: To determine the effect of triple drug immune suppression on RPE xenograft survival in the fetal pig after transplantation into the albino rabbit subretinal space. METHODS: Primary RPE microaggregates (approximately 40,000 RPE cells) were injected into the subretinal space of 24 albino rabbits, with half the rabbits maintained on triple systemic immune suppression. RPE survival was estimated with a DNA probe (porcine DNA repeat element; PRE) against a porcine-specific repetitive chromosomal marker or a RAM-11 antibody against rabbit macrophages. RESULTS: Numerous pigmented cells were visible in the subretinal space at all time points, but most pigment-containing cells 4 weeks or more after surgery were RAM-11 positive and PRE negative. The number of PRE-positive cells in the immune-suppressed group (4193 +/- 2461, 1184 +/- 1502, and 541 +/- 324 at 4, 8, and 12 weeks, respectively) was greater than in immune-competent control animals (292 +/- 506, 193 +/- 173, and 111 +/- 96), but the difference was only statistically significant at 4 weeks. The time-dependent decrease in PRE-positive cells was more pronounced in immune-suppressed animals. Image analysis performed on serial fundus photographs and fluorescein angiograms did not detect any difference in the appearance of the grafts in immune-suppressed versus immune-competent animals. CONCLUSIONS: Systemic immune suppression increased the 4-week survival of porcine RPE xenografts in the albino rabbit subretinal space, but there was poor survival in immune-suppressed and -competent animals 12 weeks after surgery. Many pigment-containing cells 4 or more weeks after surgery were PRE negative, indicating that they are of host origin.

Potent pleiotropic anti-angiogenic effects of TM601, a synthetic chlorotoxin peptide.

UNLABELLED: Chemically synthesized chlorotoxin (TM601) has been studied as a tumor targeting peptide. In this study, the anti-angiogenic properties of TM601 are reported. MATERIALS AND METHODS: In vitro and in vivo models of angiogenesis and tumor growth were used to characterize the anti-angiogenic effects of TM601. RESULTS: TM601 bound to proliferating vascular endothelial cells, decreased human umbilical vein endothelial cell (HUVEC) invasion, and reduced secretion of bioactive matrix metalloproteinase-2 (MMP-2). Using the chick chorioallantoic membrane assay (CAM), TM601 inhibited angiogenesis stimulated by any of eight pro-angiogenic factors, and when TM601 was co-administered with bevacizumab, the combination was significantly more potent than a ten-fold increase in bevacizumab dose. TM601 did not alter tumor or vascular endothelial cell growth in vitro, but TM601 treatment of tumors grown on the CAM decreased tumor growth and intra-tumoral hemoglobin levels. Intravenously injected TM601 was also shown to significantly decrease new blood vessel growth in mice. CONCLUSION: TM601 inhibits angiogenesis stimulated by many factors and potentiates the anti-angiogenic effect of bevacizumab.

Correlation of autologous skeletal myoblast survival with changes in left ventricular remodeling in dilated ischemic heart failure.

OBJECTIVES: The effect of autologous skeletal myoblast transplantation has not been rigorously studied in the setting of end-stage ischemic heart failure free of concomitant coronary revascularization. The aims of the present study were to determine autologous skeletal myoblast survival and its effects on left ventricular function and remodeling in sheep with dilated ischemic heart failure. METHODS: Ischemic heart failure (left ventricular ejection fraction, 30% +/- 2%; left ventricular end-systolic volume index, 82 +/- 9 mL/m2) was created in sheep (n = 11) with serial left circumflex coronary artery microembolizations. Instruments were inserted for the long-term determination of left ventricular global and regional dimensions, hemodynamics, and pressure-volume analysis after autologous skeletal myoblast transplantation (approximately 3.0 x 10(8) myoblasts; heart failure plus autologous skeletal myoblast group, n = 5) or without (heart failure-control group, n = 6). Measurements were performed in conscious animals. RESULTS: Autologous skeletal myoblast-derived skeletal muscle was found in all injected animals at 6 weeks. In ischemic heart failure, autologous skeletal myoblast cardiomyoplasty failed to improve systolic (left ventricular ejection fraction, 29% +/- 4%; dP/dT(max), 2863 +/- 152 mm Hg/s; end-systolic elastance, 1.6 +/- 0.22) or diastolic (left ventricular end-diastolic pressure, 21 +/- 2 mm Hg; time constant of relaxation (Tau), 34 +/- 4 ms; dP/dT(min), -1880 +/- 68 mm Hg/s) function. There was, however, attenuation in the left ventricular dilatation after autologous skeletal myoblast transplantation (change in end-systolic volume index, 14% +/- 4% vs 32% +/- 6%; P < .05). The effects of autologous skeletal myoblast-derived skeletal muscle were exclusive to the left ventricular short-axis dimension and dependent on autologous skeletal myoblast survival (R2 = 0.59, P = .006, n = 11). CONCLUSIONS: Autologous skeletal cardiomyoplasty was able to attenuate left ventricular remodeling in sheep with end-stage ischemic heart failure.

Comparison of fresh and cryopreserved porcine ventral mesencephalon cells transplanted in A rat model of Parkinson's disease.

To evaluate whether cryopreservation of porcine ventral mesencephalon cells influences graft survival and function in vivo, we have transplanted either freshly prepared or cryopreserved cells into the striatum of 6-hydroxydopamine-lesioned rats. A single cell suspension of porcine ventral mesencephalon cells from the same isolation either was stored at 4 degrees C and transplanted the next day or was cryopreserved for 4 weeks in liquid nitrogen vapor. The cryopreserved cells were then rapidly thawed, rinsed, and transplanted in the same manner as the fresh cells, with the same dose of viable cells. All animals received daily injections of cyclosporin A to prevent xenograft rejection. To monitor graft function, amphetamine-induced rotation was measured every 3 weeks between 6 and 15 weeks posttransplantation. After sacrifice at 15 weeks posttransplantation, histological methods were used to compare fresh cell and cryopreserved cell transplants with respect to graft survival, differentiation and integration, and host immune response. Cryopreserved cells were found to be either equivalent or in some cases superior to fresh cells with respect to rotational correction, graft survival, graft volume, numbers of graft-derived dopaminergic neurons, and host immune responses. In conclusion, the results indicate that it is feasible to cryopreserve porcine ventral mesencephalon cells for long-term storage of cells prior to transplantation in an animal model of Parkinson's disease.