AAV gene therapy vectors in the TMJ.

OBJECTIVES: The goal of this project was to evaluate the use of two adeno-associated viral vector serotypes, adeno-associated viral vectors (AAV)-2 and AAV-6, approved for and used for gene therapy in humans, for the delivery of therapeutic genes to the temporomandibular joint (TMJ) and the attendant sensory nerves. METHODS: Young adult wild-type C57BL/6 mice were intra-articularly inoculated with AAV-2 and AAV-6 encoding the reporter gene gfp, the expression of which was assessed in the TMJ as well as along nerves innervating the TMJ. RESULTS: AAV-2 and AAV-6 serotypes were characterized by varying levels of tissue tropism demonstrating different efficacy of infection for articular chondrocytes, meniscal fibroblasts, and trigeminal neurons. Specifically, AAV-2 infected both neurons and articular chondrocytes/meniscal fibroblasts, whereas AAV-6 showed selectivity primarily for neurons. CONCLUSIONS: The results of this study are clinically significant in the successful application of gene therapy vectors for TMJ disorders, as this new knowledge will allow for appropriate targeting of specific therapeutic genes to selective tissues (neurons vs. chondrocytes/fibroblasts) as needed by using specific viral vector serotypes.

Conditional ablation of E-cadherin in the oral epithelium progeny results in tooth anomalies.

OBJECTIVES: The objective of this study is to confirm the developmental origin of the enamel organ and evaluate the role of E-cadherin in tooth development by conditional deletion in the oral epithelium and its enamel organ progeny. K5-Cre;ROSA26 compound mice were included in this study in order to confirm the oral epithelial origin of the enamel organ, as well as of the action of the K5-Cre transgene in ablating E-cadherin in the enamel organ. K5-Cre;Ecadfl/fl knockout mice were included to evaluate the effects of the conditional E-cadherin ablation onto tooth development. MATERIAL AND METHODS: K5-Cre transgenic mice were crossed into the ROSA26 reporter mouse to trace the cell fate of the oral epithelium and its progeny in vivo. Moreover, K5-Cre mice were crossed into the Ecadfl/fl mice to produce K5-Cre;Ecadfl/fl compound mouse, as well as K5-Cre;Ecadfl/+ and Ecadfl/fl littermate controls. These litters were euthanized at postnatal day P2 to study the effects of conditional E-cadherin ablation in vivo. RESULTS: The K5-Cre;ROSA26 compound mouse demonstrated that the origin of the enamel organ and the structures thereof are of oral epithelial origin. Furthermore, using the K5-Cre;Ecadfl/fl compound mouse, we determined that conditional ablation of E-cadherin in the oral epithelium, and its progeny, results in dental anomalies involving elongation of the molar root, shrinkage of the pulp space, and alterations of the periapical area, including cementum hyperplasia. The K5-Cre;Ecadfl/fl mice also displayed a smaller overall stature compared with heterozygotes and wild-type littermates. CONCLUSIONS: E-cadherin is important in tooth development, including the formation of enamel, the crown, pulp space, and the roots.

Calcitonin gene-related peptide: An intra-articular therapeutic target for TMJ disorders.

OBJECTIVES: The goal of this project was to evaluate the role of calcitonin gene-related peptide (CGRP) in the development of arthritis. METHODS: Herein, we employed somatic mosaic analysis in two different joints by FIV(CGRP) intra-articular inoculation in the knees or temporomandibular joints (TMJ) of young adult male C57/BL6 mice. FIV(CGRP) is a feline immunodeficiency virus over-expressing full-length CGRP. Joint pathology and function were evaluated at the histopathological and behavioral levels. In addition, CGRP signaling was inhibited by intra-articular inoculation using FIV(CGRP8-37 ), such that the inhibitory peptide CGRP(8-37) was overexpressed 4 weeks after induction of joint inflammation in the TMJ of IL-1ßXAT transgenic mouse model. The mice were evaluated for behavior and killed for evaluation of knee and TMJ pathology. RESULTS: Overexpression of CGRP in the joints of wild-type mice induced the development of joint anomalies, including meniscal hypertrophy and articular pathology, associated with nocifensive behavior. Intriguingly, overexpression of the CGRP(8-37) inhibitory peptide in the knee and TMJ of IL-1ßXAT transgenic mice with joint inflammation resulted in partial amelioration of the attendant joint pathology. CONCLUSIONS: The results of this study suggest that CGRP is sufficient and necessary for the development of joint pathology and may serve as an intra-articular therapeutic target using gene therapy or monoclonal antibody-based therapies.

Partial epithelial-mesenchymal transition during enamel development.

OBJECTIVES: We set out to investigate whether a hybrid stem-like p-EMT phenotype develops during murine molar enamel development in vivo. SETTING AND SAMPLE POPULATION: Histology specimens incorporating molar tooth buds harvested from mice at post-natal day 4 (P4) were included in our studies. MATERIALS AND METHODS: We employed double immunofluorescence staining to analyze the simultaneous expression of the epithelial marker E-cadherin and the mesenchymal marker N-cadherin in histology specimens with tooth buds harvested from P4 mice. Moreover, we evaluated the expression of the core master stem cell markers Oct4 and Sox2, as well as the mature ameloblast marker amelogenin. RESULTS: Here we document the co-expression of E-cadherin and N-cadherin in a sub-population of pre-ameloblasts in the inner enamel epithelium suggestive of the presence of a hybrid epithelial/mesenchymal phenotype resulting from p-EMT. Moreover, the core stem cell factors Oct4 and Sox2 colocalized with E-cadherin expressing pre-ameloblasts, whereas the mesenchymal marker N-cadherin was expressed specifically by amelogenin-positive mature secretory ameloblasts. CONCLUSIONS: The differentiation of E-cadherin-positive pre-ameloblasts towards N-cadherin-positive mature secretory ameloblasts transition through a previously unidentified epithelial/mesenchymal stage derived through p-EMT, co-expressing the master transcription factors Oct4 and Sox2.

Reprogramming oral epithelial keratinocytes into a pluripotent phenotype for tissue regeneration.

OBJECTIVES: We set out to reprogram adult somatic oral epithelial keratinocytes into pluripotent cells for regenerative dentistry. SETTING AND SAMPLE POPULATION: Immortalized murine oral keratinocyte cell (IMOK) line raised from adult mouse mucosa were cultured in vitro in our studies. MATERIALS AND METHODS: Adult murine oral epithelial keratinocytes were chronically treated with TGF-ß1 in vitro, and the expression of Oct4, Nanog, Sox2 and Nestin, as well as specific homeobox Gata and Pax gene family members were investigated. RESULTS: We documented the induction of stem factors linked with pluripotency and/or the maintenance and regulation of stem-cell self-renewal in oral epithelial keratinocytes by TGFß1. Moreover, we discovered that this TGF-ß1-induced increase in Oct4, Nanog, Sox2 and Nestin was inhibited by SB431542, suggesting that TGF-ß1 signals via the TGF-ßRI receptor to induce pluripotency and stemness. CONCLUSIONS: Adult oral epithelial keratinocytes treated chronically with TGF-ß1 acquired phenotypic characteristics consistent with pluripotent stem cells, highlighting the facileness of reprogramming adult oral keratinocytes into an unlimited supply of pluripotent stem cells.

Serotonin contributes to the in vitro production of a biomimetic enamel-like material from reprogrammed oral epithelial keratinocytes.

OBJECTIVES: To evaluate the role of serotonin in the development of a biomimetic enamel-like material in vitro. SETTING AND SAMPLE POPULATION: Immortalized murine oral keratinocytes raised from adult mouse mucosa were cultured in vitro. In addition, specimens incorporating molar tooth buds harvested from mice were included in our studies. MATERIALS AND METHODS: We used cell-based scaffold-free tissue engineering to assemble three-dimensional (3D) organoids into complex tissue constructs that closely emulate the complexity of adult enamel. We also analysed mouse molar specimens using immunohistochemistry for serotonin expression as well as its cognate receptor. RESULTS: TGF-ß1-reprogrammed murine oral keratinocytes formed organoids that laid down an amelogenin-rich protein matrix when grown as three-dimensional (3D) cultures in the presence of serotonin. Following mineralization, the newly formed crystals were densified under pressure and vacuum to produce a biomimetic enamel-like material that demonstrated parallel alignment of hydroxyapatite crystals with some interspaced residual organoid matter into enamel prism-like structures conferring size, mechanical properties comparable to tooth enamel, including light translucency. Serotonin expression was localized by immunohistochemistry proximal to the enamel organ of developing molar buds. Moreover, serotonin HTRb2 receptor expression was localized on ameloblasts within the enamel organ proximal to the area where serotonin was immunolocalized. CONCLUSIONS: Our results demonstrate that serotonin is inductive in the development of a biomimetic enamel-like material from reprogrammed oral epithelial keratinocytes in vitro. The facileness of harvesting adult somatic cells together with the versatility of our approach offers exciting opportunities to address regenerative challenges linked to lost enamel.

Ectodomain-specific E-cadherin antibody suppresses skin SCC growth and reduces tumor grade: a multitargeted therapy modulating RTKs and the PTEN-p53-MDM2 axis.

Tumor cell survival consists of an intricate balance between cell growth and cell death pathways involving receptor tyrosine kinases [RTK; i.e., HER1-4, insulin-like growth factor-1 receptor (IGF-1R), etc.], MDM2, and the tumor suppressor proteins phosphatase and tensin homolog deleted on chromosome ten (PTEN) and p53. We recently demonstrated that shedded E-cadherin extracellular domain fragment (sEcad) is a valid oncogenic target that is significantly increased in human clinical skin squamous cell cancers (SCC) samples, UV-induced mouse tumors, and cells and promotes tumor cell proliferation, migration, and invasion by interacting and activating with the HER-phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK) axis. In resected human SCC tumors, we reported enhanced sEcad-HER1, sEcad-HER2, and sEcad-IGF-1R, but not FL-Ecad-RTK interactions. Here, we demonstrate that a sEcad antibody against the ectodomain of E-cadherin suppressed SCC growth and increased tumor differentiation in orthotopic cutaneous SCC xenografts by inhibiting proliferation and inducing apoptosis. A similar anti-sEcad antibody-induced inhibition of proliferation and induction of cell death was evident in PAM212 cells in vitro. Mechanistically, anti-sEcad administration upregulated an array of cell death pathways (i.e., Bad, active caspase-3, and cleaved PARP) and inhibited inhibitors of apoptosis (IAP; survivin, livin, etc.), RTKs (HER1, HER2, p95HER2, and IGF-1R), MAPK and PI3K/mTOR prosurvival signaling. Interestingly, in anti-sEcad mAb-treated tumors and PAM212 cells, this effect was associated with a profound increase in membrane, cytosolic, and nuclear levels of PTEN; enhanced cytosolic p53; and a decrease in MDM2 levels. Overall, our studies suggest that an antibody-based therapy against sEcad may be a novel therapeutic platform for cutaneous SCCs by hampering key proto-oncogenes (RTKs, IAPs, and MDM2) and activating potent tumor suppressor proteins (PTEN and p53) intricately linked to tumor growth and survival.

Soluble-E-cadherin activates HER and IAP family members in HER2+ and TNBC human breast cancers.

Recent literature suggests that sEcad exerts pro-oncogenic effects, possibly acting as a ligand for the human epidermal growth factor family. Here we show that sEcad is a novel candidate protein for drug targeting since it is increased in human and mouse HER2-positive (HER2+) breast tumors, MMTV-PyMT bodily fluids and human cell culture systems. Mechanistically, we show that endogenous sEcad, and to a lesser extent membrane-bound E-cadherin, associates with HER1, HER2, and HER3 in human and MMTV-PyMT mouse HER2+ tumors and with HER1 in triple negative breast cancer (TNBC) specimens. Furthermore, addition of exogenous recombinant human E-cadherin/Fc chimeric protein (rhEcad/Fc; sEcad) to HER2+ MCF-7, SKBR3, and HER2-negative MDA-MB-231 TNBC cells, resulted in sEcad-HER receptor family interactions, activation of HER1-4 and downstream pro-survival signaling, including the MAPK-PI3K/Akt/mTOR pathways and IAP family members. Lastly, we demonstrate that sEcad exerts pro-oncogenic effects via HER signaling, and acts additively with the HER ligand EGF to promote HER2+ breast cancer proliferation and migration, as well as TNBC invasion. Because sEcad associates and activates many of the oncogenic pathways that tumors utilize for growth and survival and serum levels in patients correlates with clinical response, suggests that targeted therapy against sEcad in combination with other therapies may potentially offer a novel therapeutic strategy for the treatment of breast cancers.

Monoclonal antibody against the ectodomain of E-cadherin (DECMA-1) suppresses breast carcinogenesis: involvement of the HER/PI3K/Akt/mTOR and IAP pathways.

PURPOSE: Although targeted therapies against HER2 have been one of the most successful therapeutic strategies for breast cancer, patients eventually developed acquired resistance from compensatory upregulation of alternate HERs and mitogen-activated protein kinase-phosphoinositide 3-kinase (PI3K)/Akt/mTOR signaling. As we and others have shown that the soluble ectodomain fragment of E-cadherin exerts prooncogenic effects via HER1/2-mediated binding and activation of downstream prosurvival pathways, we explored whether targeting this ectodomain [DECMA-1 monoclonal antibody (mAb)] was effective in the treatment of HER2-positive (HER2(+)) breast cancers. EXPERIMENTAL DESIGN: MMTV-PyMT transgenic mice and HER2(+)/E-cadherin-positive MCF-7 and BT474 trastuzumab-resistant (TtzmR) cells were treated with the DECMA-1 mAb. Antitumor responses were assessed by bromodeoxyuridine incorporation, apoptosis, and necrosis. The underlying intracellular prooncogenic pathways were explored using subcellular fractionation, immunoprecipitation, fluorescence microscopy, and immunoblotting. RESULTS: Treatment with DECMA-1 mAb significantly delayed tumor onset and attenuated tumor burden in MMTV-PyMT mice by reducing tumor cell proliferation and inducing apoptosis without any detectable cytotoxicity to mice or end-organs. In vitro treatment of MCF-7 and BT474 TtzmR cells reduced proliferation and induced cancer cell apoptosis. Importantly, this inhibition of breast tumorigenesis was due to concomitant downregulation, via ubiquitin-mediated degradation through the lysosome and proteasome pathways, of all HER family members, components of downstream PI3K/Akt/mTOR prosurvival signaling and suppression of inhibitor of apoptosis proteins. CONCLUSIONS: Our results establish that the E-cadherin ectodomain-specific mAb DECMA-1 inhibits Ecad(+)/HER2(+) breast cancers by hindering tumor growth and inducing apoptosis via downregulation of key oncogenic pathways involved in trastuzumab resistance, thereby establishing a novel therapeutic platform for the treatment of HER2(+) breast cancers.

Conditional expression of human ß-hexosaminidase in the neurons of Sandhoff disease rescues mice from neurodegeneration but not neuroinflammation.

This study evaluated whether GM(2) ganglioside storage is necessary for neurodegeneration and neuroinflammation by performing ß-hexosaminidase rescue experiments in neurons of HexB(-/-) mice. We developed a novel mouse model, whereby the expression of the human HEXB gene was targeted to neurons of HexB(-/-) mice by the Thy1 promoter. Despite ß-hexosaminidase restoration in neurons was sufficient in rescuing HexB(-/-) mice from GM(2) neuronal storage and neurodegeneration, brain inflammation persisted, including the presence of large numbers of reactive microglia/macrophages due to persisting GM(2) presence in this cell type. In conclusion, our results suggest that neuroinflammation is not sufficient to elicit neurodegeneration as long as neuronal function is restored.

Osteoarthritis accelerates and exacerbates Alzheimer's disease pathology in mice.

BACKGROUND: The purpose of this study was to investigate whether localized peripheral inflammation, such as osteoarthritis, contributes to neuroinflammation and neurodegenerative disease in vivo. METHODS: We employed the inducible Col1-IL1ßXAT mouse model of osteoarthritis, in which induction of osteoarthritis in the knees and temporomandibular joints resulted in astrocyte and microglial activation in the brain, accompanied by upregulation of inflammation-related gene expression. The biological significance of the link between peripheral and brain inflammation was explored in the APP/PS1 mouse model of Alzheimer's disease (AD) whereby osteoarthritis resulted in neuroinflammation as well as exacerbation and acceleration of AD pathology. RESULTS: Induction of osteoarthritis exacerbated and accelerated the development of neuroinflammation, as assessed by glial cell activation and quantification of inflammation-related mRNAs, as well as Aß pathology, assessed by the number and size of amyloid plaques, in the APP/PS1; Col1-IL1ßXAT compound transgenic mouse. CONCLUSION: This work supports a model by which peripheral inflammation triggers the development of neuroinflammation and subsequently the induction of AD pathology. Better understanding of the link between peripheral localized inflammation, whether in the form of osteoarthritis, atherosclerosis or other conditions, and brain inflammation, may prove critical to our understanding of the pathophysiology of disorders such as Alzheimer's, Parkinson's and other neurodegenerative diseases.

The trigeminal retrograde transfer pathway in the treatment of neurodegeneration.

The trigeminal sensory system was evaluated for the retrograde transfer of gene therapy vectors into the CNS. The feline immunodeficiency viral vector, FIV(HEXB), encoding for the human HEXB gene, was injected intra-articularly in the temporomandibular joint of 12 week-old HexB(-/-) mice displaying clinical and histopathological signs of Sandhoff disease. This treatment regiment reduced GM(2) storage and ameliorated neuroinflammation in the brain of HexB(-/-) mice, as well as attenuated behavioral deficits. In conclusion, retrograde transfer along trigeminal sensory nerves may prove to be a valuable route of gene therapy administration for the treatment of lysosomal storage disorders and other neurodegenerative diseases.

Spinal interleukin-1beta in a mouse model of arthritis and joint pain.

OBJECTIVE: Pain from arthritis has been associated with peripheral sensitization of primary sensory afferents and the development of inflammation at the dorsal horns. This study was undertaken to determine whether the role of spinal interleukin-1beta (IL-1beta) in central processing of pain is important in the development of arthritis. METHODS: Col1-IL-1betaXAT mice and GFAP-IL-1betaXAT mice were injected with the feline immunodeficiency virus (FIV) (Cre) vector in the right and left temporomandibular joints (TMJs), or in the cisterna magna, respectively, to induce IL-1beta expression in the dorsal horns of the spinal horn. To inhibit intrathecal IL-1 receptor type I (IL-1RI) signaling, FIV(IL-1Ra) vector was injected into the cisterna magna of Col1-IL-1betaXAT mice. The effects of IL-1RI receptor inhibition in GFAP-IL-1betaXAT mice were studied in the GFAP-IL-1betaXAT-IL-1RI(-/-) compound mouse model. Neuroinflammatory, sensory, and behavioral changes were evaluated in conjunction with arthritic changes in the TMJ, assessed by histopathologic and immunohistochemical analyses. RESULTS: Induction of an osteoarthritis-like condition in the TMJ in the Col1-IL-1betaXAT mouse model resulted in up-regulation of murine IL-1beta at the dorsal horns. Moreover, intrathecal inhibition of IL-1RI in Col1-IL-1betaXAT mice with arthritis led to amelioration of joint pathology and attenuation of the attendant joint pain. Overexpression of spinal IL-1beta in the recently developed GFAP-IL-1betaXAT somatic mosaic model of neuroinflammation led to development of arthritis-like pathology accompanied by increased pain-like behavior. CONCLUSION: Our results indicate that joint pathology and pain are dependent on spinal IL-1beta, and suggest the presence of a bidirectional central nervous system-peripheral joints crosstalk that may contribute to the development, expansion, and exacerbation of arthritis.

Peripheral blood mononuclear cell infiltration and neuroinflammation in the HexB-/- mouse model of neurodegeneration.

Myeloid-derived immune cells, including microglia, macrophages and monocytes, have been previously implicated in neurodegeneration. We investigated the role of infiltrating peripheral blood mononuclear cells (PBMC) in neuroinflammation and neurodegeneration in the HexB-/- mouse model of Sandhoff disease. Ablation of the chemokine receptor CCR2 in the HexB-/- mouse resulted in significant inhibition of PBMC infiltration into the brain, decrease in TNFalpha and MHC-II mRNA abundance and retardation in clinical disease development. There was no change in the level of GM2 storage and pro-apoptotic activity or astrocyte activation in HexB-/-; Ccr2-/- double knockout mice, which eventually succumbed secondary to GM2 gangliosidosis.

Intraperitoneal inoculation of Sandhoff mouse neonates with an HIV-1 based lentiviral vector exacerbates the attendant neuroinflammation and disease phenotype.

We aimed to evaluate the efficacy of VSV-G pseudotyped, defective HIV-1 based lentiviral vectors for the neonatal transfer of therapeutic genes following systemic administration in Sandhoff mouse pups. Despite transgene expression in mouse brains, these animals presented with significant exacerbation and acceleration of the disease neurological phenotype. We observed an increase and acceleration in the presence of MHC-II and CD45+ cells in their brains, along with neuroinflammation, but not in control heterozygous or wild type littermates that also received the same treatment.

beta-hexosaminidase lentiviral vectors: transfer into the CNS via systemic administration.

Brain inflammation in GM2 gangliosidosis has been recently realized as a key factor in disease development. The aim of this study was to investigate the effects of a FIV beta-hexosaminidase vector in the brain of HexB-deficient (Sandhoff disease) mice following intraperitoneal administration to pups of neonatal age. Since brain inflammation, lysosomal storage and neuromuscular dysfunction are characteristics of HexB deficiency, these parameters were employed as experimental outcomes in our study. The ability of the lentiviral vector FIV(HEX) to infect murine cells was initially demonstrated with success in normal mouse fibroblasts and human Tay-Sachs cells in vitro. Furthermore, systemic transfer of FIV(HEX) to P2 HexB-/- knockout pups lead to transduction of peripheral and central nervous system tissues. Specifically, beta-hexosaminidase expressing cells were immunolocalized in periventricular areas of the cerebrum as well as in the cerebellar cortex. FIV(HEX) neonatal treatment resulted in reduction of GM2 storage along with attenuation of the brain inflammation and amelioration of the attendant neuromuscular deterioration. In conclusion, these results demonstrate the effective transfer of a beta-hexosaminidase lentiviral vector to the brain of Sandhoff mice and resolution of the GM2 gangliosidosis after neonatal intraperitoneal administration.