Gregory A. Helm

In vivo endochondral bone formation using a bone morphogenetic protein 2 adenoviral vector

Bone morphogenetic proteins (BMPs) are polypeptides that induce ectopic bone formation in standard rat in vivo assay systems. Previous studies have demonstrated the clinical utility of these proteins in spinal fusion, fracture healing, and prosthetic joint stabilization. Gene therapy is also a theoretically attractive technique to express BMPs clinically, since long-term, regulatable gene expression and systemic delivery with tissue-specific expression may be possible in future. This study was performed to determine whether an adenoviral vector containing the BMP-2 gene can be used to express BMP-2 in vitro and promote endochondral bone formation in vivo. In vitro, U87 MG cells transduced per cell with 20 MOI of an adenoviral construct containing the BMP-2 gene under the control of the universal CMV promoter (Ad-BMP-2) showed positive antibody staining for the BMP-2 protein at posttransfection day 2. The synthesis and secretion of active BMP-2 into the conditioned medium of Ad-BMP-2-transduced 293 cells were confirmed by Western blot analysis and the induction of alkaline phosphatase activity in a W-20 stromal cell assay. In vivo, Sprague-Dawley rats and athymic nude rats were injected with Ad-BMP-2 in the thigh musculature and were sacrificed on day 3, 6, 9, 12, 16, 21, 60, and 110 for histological analysis. The Sprague-Dawley rats showed evidence of acute inflammation, without ectopic bone formation, at the injection sites. In the athymic nude rats, BMP-2 gene therapy induced mesenchymal stem cell chemotaxis and proliferation, with subsequent differentiation to chondrocytes. The chondrocytes secreted a cartilaginous matrix, which then mineralized and was replaced by mature bone. This study demonstrates that a BMP-2 adenoviral vector can be utilized to produce BMP-2 by striated muscle cells in athymic nude rats, leading to endochondral bone formation. However, in immunocompetent animals the endochondral response is attenuated, secondary to the massive immune response elicited by the first-generation adenoviral construct.

Osteogenic potential of five different recombinant human bone morphogenetic protein adenoviral vectors in the rat.

Bone morphogenetic protein (BMP) adenoviral vectors for the induction of osteogenesis are being developed for the treatment of bone pathology. However, it is still unknown which BMP adenoviral vector has the highest potential to stimulate bone formation in vivo. In this study, the osteogenic activities of recombinant human BMP-2, BMP-4, BMP-6, BMP-7, and BMP-9 adenoviruses were compared in vitro, in athymic nude rats, and in Sprague–Dawley rats. In vitro osteogenic activity was assessed by measuring the alkaline phosphatase activity in C2C12 cells transduced by the various BMP vectors. The alkaline phosphatase activity induced by 2 × 105 PFU/well of BMP viral vector was 4890 × 10−12 U/well for ADCMVBMP-9, 302 × 10−12 U/well for ADCMVBMP-4, 220 × 10−12 U/well for ADCMVBMP-6, 45 × 10−12 U/well for ADCMVBMP-2, and 0.43 × 10−12 U/well for ADCMVBMP-7. The average volume of new bone induced by 107 PFU of BMP vector in athymic nude rats was 0.37±0.03 cm3 for ADCMVBMP-2, 0.89±0.07 cm3 for ADCMVBMP-4, 1.02±0.07 cm3 for ADCMVBMP-6, 0.24±0.05 cm3 for ADCMVBMP-7, and 0.63±0.07 cm3 for ADCMVBMP-9. In immunocompetent Sprague–Dawley rats, no bone formation was demonstrated in the ADCMVBMP-2, ADCMVBMP-4, and ADCMVBMP-7 groups. ADCMVBMP-6 at a viral dose of 108 PFU induced 0.10±0.03 cm3 of new bone, whereas ADCMVBMP-9 at a lower viral dose of 107 PFU induced more bone, with an average volume of 0.29±0.01 cm3.

Impaired axonal transport and altered axolemmal permeability occur in distinct populations of damaged axons following traumatic brain injury.

Traumatic axonal injury (TAI) evolves within minutes to hours following traumatic brain injury (TBI). Previous studies have identified axolemmal disruption and impaired axonal transport (AxT) as key mechanisms in the evolution of TAI. While initially hypothesized that axolemmal disruption culminates in impaired AxT, previous studies employed single-label methodologies that did not allow for a full determination of the spatial-temporal relationships of these two events. To explore directly the relationship between impaired AxT and altered axolemmal permeability, the current investigation employed 40, 10, and 3 kDa fluorescently conjugated dextrans as markers of axolemmal integrity, with antibodies targeting the anterogradely transported amyloid precursor protein (APP) utilized as a marker of impaired AxT. Rats underwent impact acceleration TBI and were intrathecally administered 40 kDa, 40 + 10 kDa or 40 + 3 kDa fluorescently tagged dextrans, with brains subsequently prepared for APP immunofluorescence. Brainstem corticospinal tracts (CSpT), medial lemnisci (ML), and medial longitudinal fasciculi were examined for evidence of TAI. APP and all dextrans consistently localized to distinct classes of TAI. Dextrans were noted as early as 5 min following injury within axonal segments demonstrating an irregular/tortuous appearance, and were seen within thin and elongate/vacuolated axons by 30 min-6 h following injury. APP, first noted within swollen axons at 30 min following injury, was found within progressively swollen axons that showed no dextran colocalization within 3 h of injury. However, by 6 h, dextrans colocalized in disconnected axonal bulbs. At this time-point, dextrans also persisted within single-labeled, highly vacuolated/thin, and elongate axons. These studies confirm that axolemmal disruption and impaired AxT occur as distinct non-related events early in the pathogenesis of TAI. Further, these studies provide evidence that the process of impaired axonal transport and subsequent axonal disconnection leads to delayed axolemmal instability, rather than proceeding as a consequence of initial axolemmal failure. This finding underscores the need of multiple approaches to fully assess the axonal response to TBI.

Expression of survivin, an inhibitor of apoptosis protein, in tumors of the nervous system

Abstract. Survivin is an inhibitor of apoptosis protein that blocks apoptosis by binding to caspases-3 and -7. It is highly expressed in less-differentiated embryonic cells and rapidly dividing tumors, but not in terminally differentiated adult tissues. Elevated survivin levels are found in malignant systemic tumors, and are associated with chemo-resistance, radiation resistance, and poor prognosis. However, expression of survivin in primary nervous system tumors has not been previously characterized. Immunohistochemistry using anti-human survivin antibody (SURV11-A) was performed on formalin-fixed, paraffin-embedded archival tissue from 112 primary central nervous system tumors. Survivin immunoreactivity was seen in most diffuse astrocytomas [WHO II (2/4), III (3/3), IV (9/10), giant-cell glioblastoma (1), and gliosarcoma (1)]. The intensity and degree of survivin expression showed trends with tumor grade, with glioblastomas having the highest positivity. Pilocytic astrocytomas (5) and pleomorphic xanthoastrocytoma (1) were positive to a lesser degree. In oligodendrogliomas (6) and mixed oligo-astrocytomas [grade II (5), II–III (3), and III (7)], oligodendroglial elements appear to be negative compared to positive mini-gemistocytic oligodendrocytes. Ependymomas [grade II (6) and grade III (1)] were positive. Medulloblastomas (5) and retinoblastoma (1/4) showed focal positivity. All meningiomas [grade I (12), II (9), III (4), and grade I (3) and II (5) with frank brain invasion] were intensely positive. All schwannomas (11) and neurofibromas (6) were intensely positive. Thus, survivin is expressed in the majority of the primary nervous system tumors, particularly in glioblastomas, meningiomas, schwannomas and neurofibromas. Overexpression of survivin in meningiomas and benign peripheral nerve sheath tumors contrasts with previous reports relating it to rapid division and poor prognosis.

The use of bone morphogenetic protein gene therapy in craniofacial bone repair.

&NA; Bone morphogenetic proteins (BMPs) are capable of inducing endochondral bone formation when applied on biologic carriers in numerous mammalian in vivo assay systems. Bone morphogenetic protein gene therapy is also currently being developed to promote osteogenesis for clinical indications such as spinal fusions, craniofacial bone loss, and osteoporosis. In this study, critical‐sized mandibular defects were treated with a control adenoviral vector (Ad‐&bgr;‐gal), a BMP‐2 adenoviral vector (Ad‐BMP‐2), or a BMP‐9 adenoviral vector (Ad‐BMP‐9). Gross tissue examination, radiographic analysis, and histologic analysis demonstrated significant bony healing in the BMP treated groups compared to controls. Osteogenesis was limited to the bony defect, without extension into the surrounding soft tissues. The study suggests that with further development, BMP gene therapy may be potentially useful for repair of bony defects in the craniofacial region.

Ex vivo bone morphogenetic protein-9 gene therapy using human mesenchymal stem cells induces spinal fusion in rodents.

OBJECTIVE Ex vivo gene therapy with the use of human mesenchymal stem cells (hMSCs) and bone morphogenetic protein (BMP) genes provides a local supply of precursor cells and a supraphysiological dose of osteoinductive molecules that may promote bone formation in patients with inadequate hMSC populations because of age, osteoporosis, metastatic bone disease, iatrogenic depletion, or other metabolic derangements. This study was undertaken to evaluate the efficacy of ex vivo gene therapy with the use of hMSCs and the BMP-9 gene to promote spinal fusion in the rat. METHODS Sixteen athymic nude rats were treated with hMSCs transduced with recombinant, replication-defective Type 5 adenovirus containing the cytomegalovirus promoter and either the BMP-9 (Ad-BMP-9) or the &bgr;-galactosidase (Ad-&bgr;-gal) gene. Ad-&bgr;-gal served as the control. Each animal received a percutaneous, paraspinal injection of 106 hMSCs transduced with 50 plaque-forming units/cell adenovirus in the lumbar region, with Ad-BMP-9 on the left and Ad-&bgr;-gal on the right. At 8 weeks postinjection, computed tomographic scans of the lumbosacral spine were obtained, and the lumbosacral spine specimens were examined histologically. RESULTS Both computed tomographic studies and histological analysis clearly demonstrated large volumes of ectopic bone at the Ad-BMP-9-transduced hMSC injection sites, resulting in successful spinal fusion and no evidence of nerve root compression or local or systemic toxicity. The contralateral regions that were treated with Ad-&bgr;-gal-transduced hMSCs showed no evidence of osteogenesis. CONCLUSION The results of this study suggest that hMSC and BMP-9 ex vivo gene therapy may be useful in inducing spinal fusion as well as other related procedures and certainly warrants further clinical development.

Caspase-3-Mediated Cleavage of Amyloid Precursor Protein and Formation of Amyloid β Peptide in Traumatic Axonal Injury

Immunohistochemical studies demonstrate accumulation of the beta-amyloid precursor protein (APP) within injured axons following traumatic brain injury (TBI). Despite such descriptions, little is known about the ultimate fate of accumulating APP at sites of traumatic axonal injury (TAI). Recently, caspase-3-mediated cleavage of APP and subsequent Abeta deposition was linked to apoptotic neuronal death pathways in hippocampal neurons following ischemic and excitotoxic brain injury. Given that (1) APP is known to accumulate within traumatically injured axons, (2) caspase-3 activation has been demonstrated in traumatic axonal injury (TAI), and (3) recent studies have identified a caspase-3 cleavage site within APP, we initiated the current investigation to determine whether caspase-3-mediated cleavage of APP occurs in TAI. We further assessed whether these events were found in relation to Abeta peptide formation. To this end, we employed antibodies targeting APP, the caspase-3-mediated breakdown product of APP proteolysis, and the Abeta peptide. Rats were subjected to impact acceleration TBI (6 h to 10 days survival), and their brains were processed for single-label bright field and multiple double-label immunofluorescent paradigms using the above antibodies. By 12 h postinjury, caspase-3-mediated APP proteolysis (CMAP) was demonstrated within the medial lemniscus (ML) and medial longitudinal fasciculus (MLF) in axons undergoing TAI, identified by their concomitant APP accumulation. Immunoreactivity for CMAP persisted up to 48 h postinjury in the ML and MLF, but was notably reduced by 10 days following injury. Further, CMAP was colocalized with Abeta formation in foci of TAI. The current study demonstrates that caspase-3 cleavage of APP occurs in TAI and is associated with formation of Abeta peptide. These findings are of interest given recent epidemiological studies supporting an association between TBI and later risk for AD development.

MPP+ induced apoptotic cell death in SH-SY5Y neuroblastoma cells: An electron microscope study

PD is a common, late‐onset neurodegenerative disorder that results in part from the gradual loss of dopaminergic neurons in the substantia nigra pars compacta. The neurotoxin MPTP can induce PD‐like clinical symptomatology and neuropathological destruction and, thus, has been used as a PD model. The human neuroblastoma cell line SH‐SY5Y possesses many of the qualities of human neurons and, as such, has served as a model for them. Apoptosis is the mode of cell death induced in SH‐SY5Y cells by MPTP, and this was confirmed with nick end labeling and bisbenzimide staining. Transmission electron microscopic analysis of the ultrastructural changes occurring in neurotoxin exposed SH‐SY5Ys revealed many morphological characteristics consistent with apoptosis. These changes included plasmalemmal blebbing, altered cytosolic density, nuclear condensation and fragmentation, pronounced vacuole formation, ribosomal dispersion, and the disappearance of the golgi complex, microtubules, and smooth endoplasmic reticulum. Limited amounts of rough endoplasmic reticulum and mitochondria exhibited normal morphology throughout the apoptotic changes but then were disrupted during secondary necrotic changes.

Transforming growth factor beta-coated platinum coils for endovascular treatment of aneurysms: An animal study

OBJECTIVETo test the hypothesis that coating platinum coils with transforming growth factor &bgr; (TGF&bgr;) would improve the cellular proliferation within experimental aneurysms relative to uncoated coils. MATERIALS AND METHODS Elastase-induced saccular aneurysms were created in 12 New Zealand White rabbits. These aneurysms were embolized with platinum coils, either “control” (unmodified) coils or “test” (coated with TGF&bgr;) coils. Subjects were killed either 2 weeks (n = 3, control; n = 3, test) or 6 weeks (n = 3, control; n = 3, test) after embolization. Aneurysm tissue was embedded in plastic, sectioned, and stained with hematoxylin and eosin. The thickness of tissue covering the coils at the coil-lumen interface was measured by use of a digital microscope, and was compared between groups by use of the Student’s t test (P ≤ 0.05). RESULTSTwo-week implantation samples demonstrated mean thickness of tissue overlying TGF&bgr;-coated coils of 36 ± 15 &mgr;m and mean thickness of overlying control coils of 3 ± 5 &mgr;m, indicating significantly thicker tissue growth covering test versus control coils (P = 0.02). Six-week implantation samples demonstrated mean thickness of tissue overlying TGF&bgr;-coated coils of 86 ± 74 &mgr;m versus mean thickness overlying control coils of 37 ± 6 &mgr;; this difference did not reach statistical significance (P = 0.30). Thickness of tissue covering TGF&bgr;-coated coils did not change significantly from 2 to 6 weeks (P = 0.31). Tissue thickness over control coils increased significantly between 2 and 6 weeks (P = 0.002). CONCLUSIONTGF&bgr;-coated platinum coils undergo earlier cellular coverage than standard platinum coils, but differences in coverage between coated and control coils are no longer present at later time points. These data suggest that improvements in intra-aneurysmal cellular proliferation resulting from coil modifications, although significant in the early postembolization phase, may dissipate over time.

Morphologic analysis of BMP-9 gene therapy-induced osteogenesis

The present study was performed to determine the histological, ultrastructural, and radiographic changes that occur over time at intramuscular BMP-9 gene therapy treatment sites. Several members of the bone morphogenetic protein (BMP) family have the potential to induce osteochondrogenesis when the protein is delivered to rodents, canines, rabbits, and nonhuman primates. Previous studies have also demonstrated that BMP gene therapy utilizing adenoviral vectors can also stimulate orthotopic and heterotopic bone formation in rodents and rabbits. Athymic nude and Sprague-Dawley rats were injected with Ad-BMP-9 or Ad-beta-Gal (3.75 x 10(9) particles) in their thigh musculature and light microscopic, electron microscopic, and computerized tomography analysis was performed 3, 6, 9, 12, 15, 18, 21, and 100 days later. To assess early mesenchymal cell proliferation, a bromodeoxyuridine (BrdU) immunohistochemical analysis was also performed 48, 60, and 72 hr postinjection in athymic nude rats. All animals demonstrated extensive endochondral bone formation at the Ad-BMP-9 treatment sites within 3 weeks. The Sprague-Dawley rats also exhibited a massive, acute inflammatory infiltrate during the first week. Proliferating mesenchymal stem cells were clearly evident as early as 2 days after treatment, which differentiated into small or hypertrophied chondrocytes during the next week. During the third week, the cartilaginous matrix mineralized and formed woven bone, which converted to lamellar bone by 3 months. No evidence of bone formation was demonstrated at the Ad-beta-Gal injection sites in the athymic nude or Sprague-Dawley rats. In addition, no cellular proliferation was seen at the Ad-beta-Gal treatment sites in the athymic nude animals as assessed by light microscopy and BrdU immunohistochemistry. The extensive bone formation induced by Ad-BMP-9 suggests that BMP gene therapy may have potential utility in the treatment of degenerative, rheumatic, or traumatic bone pathology.