Douglas B. Jacoby

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.

No evidence for infection of human cells with porcine endogenous retrovirus (PERV) after exposure to porcine fetal neuronal cells.

Background. Recent demonstration of human cell infection in vitro with porcine endogenous retrovirus (PERV) has raised safety concerns for new therapies that involve transplantation of pig cells or organs to humans. To assess better the specific risk that may be associated with the transplantation of fetal pig neuronal cells to the central nervous system of patients suffering from intractable neurologic disorders (Parkinson’s disease, Huntington’s disease, and epilepsy), we have performed studies to determine whether there is evidence for in vivo or in vitro transmission of PERV from fetal pig neuronal cells to human cells. Methods. Ventral mesencephalon (VM) and lateral ganglionic eminence cells were isolated from fetal pigs and transplanted into patients with neurological conditions as part of clinical studies. Blood samples taken from patients at various time points posttransplant were tested for evidence of PERV. In vitro studies to test for PERV infection of human cells after cocultivation with either fetal porcine ventral mesencephalon or porcine fetal lateral ganglionic eminence cells were also performed. Results. We found no evidence of PERV provirus integration in the DNA from PBMC of 24 neuronal transplant recipients. In addition, no PERV was released from cultured fetal porcine neuronal cultures, and there was no transfer of PERV from fetal pig neuronal cells to human cells in vitro. Conclusions. Our results demonstrate by both examination of transplant patient blood samples and in vitro studies that there is no evidence for transmission of PERV from porcine fetal neural cells to human cells.

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.

Long-term survival of fetal porcine lateral ganglionic eminence cells in the hippocampus of rats

Embryonic porcine brain tissue from the lateral ganglionic eminence was transplanted into the adult rat hippocampus to determine whether fetal striatal cells could survive, differentiate, and integrate in a heterotopic site. The hippocampus, a common site of epileptic seizure activity, was chosen to determine if fetal striatal cells could supply inhibitory GABAergic neurons that may serve to block seizures. Cells were either implanted with a single deposit using a standard metal cannula or by five smaller disseminated deposits with a glass micropipette. At 20–24 weeks, animals immunosuppressed with cyclosporin showed long‐term survival of porcine cells in the adult hippocampus. Analysis by immunohistochemistry and in situ hybridization showed that the grafts contained glial and neuronal cell types, including GABAergic neurons within graft core and networks of porcine neuronal fibers extending from the graft into the host parenchyma. In addition, a marker of porcine presynaptic terminals, synaptobrevin, was abundant within the grafts and was found associated with hippocampal structures and cell layers suggesting functional integration of grafted cells within the host. The survival of xenografts in the hippocampus and potential integration of inhibitory components provides evidence that these grafts may serve as an internal negative feedback mechanism to quench epileptiform activity. J. Neurosci. Res. 56:581–594, 1999. 

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 Bruchs 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. J. Cell. Physiol. 225: 855–864, 2010.

Regulated expression of the homeobox gene, rPtx2, in the developing rat

Using degenerate primers designed to amplify genes containing homeodomains, we have used reverse transcription and polymerase chain reaction to amplify and clone a rat homeobox gene. Based on the nucleotide and predicted amino acid sequences, the rat cDNA clone contains a high degree of sequence similarity to murine genes which are members of the paired-like class of homeobox genes (Ptx2, Otlx2, solurshin and Ptx1). Considering the high degree of sequence similarity and similar restricted expression patterns, we have named the cloned rat gene rPtx2 (rat Ptx2 homolog). Northern analysis revealed two rPtx2 transcripts expressed in the developing rat brain. Yet, only a single gene was detected by Southern blot hybridization, suggesting that multiple messages are the result of alternative transcriptional initiation, splicing or processing of a common message. The expression pattern of rPtx2 was further delineated by in situ hybridization to rat embryos. Within the brain, tissue specific expression was observed in the differentiating neural cells of the posterior hypothalamus, tegmentum, and rhombomere r1. Expression was also observed in the developing pituitary, maxilla, mandible, tongue and umbilical cord. To further study the control of Ptx2 gene expression, we used an in vitro model for neural differentiation by treating mouse embryonic stem cells with retinoic acid. Within 24 h and prior to detection of a neural phenotype in the culture, murine Ptx transcripts were induced and remained elevated for at least 6 days. This suggests that retinoic acid may be an important inductive signal which regulates the developmental and tissue-specific expression of Ptx2.

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°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 Parkinsons disease.

Identification of transplanted retinal pigment epithelium with a novel chromosomal marker

Purpose. To demonstrate the ability of a novel chromosomal marker to identify retinal pigment epithelium (RPE) after xenotransplantation, and determine the short-term correlation between pigment and this nuclear marker. Methods. Primary pigmented RPE harvested from third trimester fetal pigs were transplanted as microaggregates into the subretinal space of 3 albino rabbits. We then used an in situ probe for a repetitive segment of the porcine chromosome to identify the transplanted RPE. Results. Pigmented cells were visible in the subretinal space 2 weeks after transplantation. Approximately 70% of pigment-containing cells were also labeled with the porcine chromosomal marker. Labeled cells were predominantly flatter in morphology and close to Bruch’s membrane whereas unlabeled cells were rounder and further from Bruch’s membrane. The outer nuclear layer thickness was normal above the pigmented monolayer but was decreased over areas containing multiple layers of pigmented cells. Conclusions. Fetal porcine RPE xenografts can be identified with a nuclear marker for a repetitive segment of the porcine chromosome. The presence of pigment within unlabelled cells suggests that pigment is not a robust marker for transplanted RPE.

Abstract C89: Galeterone suppresses castration-resistant and enzalutamide-resistant prostate cancer growth in vitro.

Background: Despite the progress and innovation in chemo- and immunotherapies, androgen deprivation therapy remains the standard treatment of metastatic prostate cancer. However, progression to castrate resistance disease (CRPC) occurs in the majority of patients and intriguingly, over 80% of CRPC specimens continue to express androgen receptor (AR) and androgen-responsive genes, indicating that the AR axis remains paradoxically activated despite castration. Hence, several new classes of AR-targeting agents have been recently introduced into clinic, including more potent AR antagonists (enzalutamide). Despite the substantial clinical efficacy reported with Enzalutamide, resistance to this therapy has already been observed and reactivation of AR signaling following treatment occurs. Thus, the next major clinical challenge that we will face is identifying new therapies that prevent or treat anti-androgen resistance. Galeterone is a novel drug that exhibits three mechanisms of action to inhibit AR activity, via inhibition of de novo androgen synthesis, blocking the ligand binding domain to prevent androgen binding, and inducing AR degradation. Thus, in this study we evaluated the efficacy of this multi-potent drug to inhibit AR activity in enzalutamide resistant cells. Results: As a pre-clinical model of enzalutamide resistance, drug resistant and CRPC cell lines were derived from three generations of serially passaged enzalutamide resistant, or vehicle control treated, LNCaP xenografts. Resistant cells were maintained in vitro under constant exposure to 10 μM of enzalutamide and were used to study the anti-cancer and AR targeting effects of galeterone in the enzalutamide resistant setting. Using crystal violet and MTT assays, we found that galeterone had anti-proliferative effects in LNCaP cells, in CRPC cells, and most importantly, in those resistant to enzalutamide. Compared to enzalutamide treatment, galeterone induced a drastic decrease in Probasin luciferase reporter (AR) activity, a greater reduction in AR and prostate-specific antigen (PSA) protein expression, and the inhibition of AR nuclear translocation in all cell lines. Strikingly, these effects were still observed in resistant cells, which show no decrease in AR expression or nuclear translocation upon enzalutmide treatment. Together, our data show that galeterone strongly inhibits AR activity and suppresses castration-resistant LNCaP growth as well as enzalutamide -resistant cell growth in vitro. Conclusion: In this study, we provide a preclinical proof-of-principle that Galeterone is a potent inhibitor of the AR pathway and may represent the next generation of hormone therapy for patients with not only CRPC but also Enzalutamide resistant disease. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C89. Citation Format: Nader Al Nakouzi, Chris Wang, Douglas Jacoby, Martin E. Gleave, Amina Zoubeidi. Galeterone suppresses castration-resistant and enzalutamide-resistant prostate cancer growth in vitro. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C89.

Abstract 3490: The effect of novel CYP17 inhibitor galeterone on gonadal and tumor progestogen and androgen levels in SCID mice bearing LNCaP prostate cancer xenografts

Prostate cancer growth is driven by androgen-dependent activation of the androgen receptor (AR). Inhibition of CYP17A1, a cytochrome P450 enzyme responsible for the conversion of progestogens to androgens, is a key strategy in the treatment of prostate cancer. CYP17A1 has both 17α-hydroxylase and 17,20-lyase function. The 17α-hydroxylase catalyzes the conversion of progesterone/pregnenolone to 17α-hydroxyprogesterone/17α-hydroxypregnenolone and the 17,20-lyase converts 17α-hydroxyprogesterone/17α-hydroxypregnenolone to androstenedione (AD)/dehydroepiandrosterone (DHEA). Galeterone, a novel AR degrader and antagonist, is also an inhibitor of CYP17A1 and has been shown to inhibit the growth of prostate cancer cells and tumors. However, the effects of galeterone on steroid levels affected by CYP17A1 inhibition have not been examined in vivo. Male SCID mice bearing LNCaP tumors were randomized to receive vehicle (30% beta-cyclodextran in saline) or 0.15mmol/kg galeterone (either po or sc). Mice were treated twice daily, seven days a week. Route of administration did not alter the efficacy of galeterone, which significantly reduced tumor volume (p = 0.044 and p = 0.049, sc, po). Upon completion of the experiment, tumors, testes, and plasma were collected for analysis of steroid levels. Analysis of intratumoral steroids showed an increase in progesterone (1.7- and 2-fold, sc, po) and a decrease in AD (89% and 77% reduction, sc, po). Similar results were observed in levels of pregnenolone which increased (1.4- and 3.0-fold, sc, po) while DHEA decreased (72% and 17% reduction, sc, po). Interestingly, levels of 17α-hydroxyprenenolone were increased in the sc treatment arm, suggesting selectivity of galeterone for the lyase over the hydroxylase catalytic function. Both routes of administration reduced intratumoral testosterone (97% and 77% reduction, sc, po). Intratesticular androgen levels showed similar trends compared to those in tumors. Following galeterone treatment, the levels of androgen precursors were higher in the testes than in the tumor (pregnenolone increased 2.3- and 1.5-fold sc, po; progesterone elevated 6.4- and 12.8-fold sc, po). Plasma androgen levels varied from those observed in the tumor and testes. However, galeterone treatment reduced the levels of AD (98% and 68% reduction, sc, po) and testosterone (99.6% and 99% reduction, sc, po). Together, these results show for the first time that galeterone specifically targets CYP17A1 in vivo as demonstrated by reduction of its enzymatic products DHEA, AD and testosterone. In addition, a selectivity for the lyase function was observed, as evidenced by greater decreases in DHEA and AD than in 17α-hydroxypregenelone. This is consistent with findings that patients treated with galeterone do not require cortisol replacement therapy and do not show symptoms of mineralocorticoid excess. Citation Format: Amanda J. Schech, Gauri J. Sabnis, Stephen Yu, Vincent C.O. Njar, Douglas B. Jacoby, Peter Nelson, Ruth Dumpit, Angela M.H. Brodie. The effect of novel CYP17 inhibitor galeterone on gonadal and tumor progestogen and androgen levels in SCID mice bearing LNCaP prostate cancer xenografts. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3490.