Lynda O'Rear

Regenerative effects of transplanted mesenchymal stem cells in fracture healing.

Mesenchymal stem cells (MSC) have a therapeutic potential in patients with fractures to reduce the time of healing and treat nonunions. The use of MSC to treat fractures is attractive for several reasons. First, MSCs would be implementing conventional reparative process that seems to be defective or protracted. Secondly, the effects of MSCs treatment would be needed only for relatively brief duration of reparation. However, an integrated approach to define the multiple regenerative contributions of MSC to the fracture repair process is necessary before clinical trials are initiated. In this study, using a stabilized tibia fracture mouse model, we determined the dynamic migration of transplanted MSC to the fracture site, their contributions to the repair process initiation, and their role in modulating the injury‐related inflammatory responses. Using MSC expressing luciferase, we determined by bioluminescence imaging that the MSC migration at the fracture site is time‐ and dose‐dependent and, it is exclusively CXCR4‐dependent. MSC improved the fracture healing affecting the callus biomechanical properties and such improvement correlated with an increase in cartilage and bone content, and changes in callus morphology as determined by micro‐computed tomography and histological studies. Transplanting CMV‐Cre‐R26R‐Lac Z‐MSC, we found that MSCs engrafted within the callus endosteal niche. Using MSCs from BMP‐2‐Lac Z mice genetically modified using a bacterial artificial chromosome system to be β‐gal reporters for bone morphogenic protein 2 (BMP‐2) expression, we found that MSCs contributed to the callus initiation by expressing BMP‐2. The knowledge of the multiple MSC regenerative abilities in fracture healing will allow design of novel MSC‐based therapies to treat fractures. STEM CELLS 2009;27:1887–1898

Effect of IGF‐I in the Chondrogenesis of Bone Marrow Mesenchymal Stem Cells in the Presence or Absence of TGF‐β Signaling

A novel role for IGF‐I in MSC chondrogenesis was determined. IGF‐I effects were evaluated in the presence or absence of TGF‐β signaling by conditionally inactivating the TGF‐β type II receptor. We found that IGF‐I had potent chondroinductive actions on MSCs. IGF‐I effects were independent from and additive to TGF‐β.

TGF-β signaling is essential for joint morphogenesis

Despite its clinical significance, joint morphogenesis is still an obscure process. In this study, we determine the role of transforming growth factor β (TGF-β) signaling in mice lacking the TGF-β type II receptor gene (Tgfbr2) in their limbs (Tgfbr2PRX-1KO). In Tgfbr2PRX-1KO mice, the loss of TGF-β responsiveness resulted in the absence of interphalangeal joints. The Tgfbr2Prx1KO joint phenotype is similar to that in patients with symphalangism (SYM1-OMIM185800). By generating a Tgfbr2–green fluorescent protein–β–GEO–bacterial artificial chromosome β-galactosidase reporter transgenic mouse and by in situ hybridization and immunofluorescence, we determined that Tgfbr2 is highly and specifically expressed in developing joints. We demonstrated that in Tgfbr2PRX-1KO mice, the failure of joint interzone development resulted from an aberrant persistence of differentiated chondrocytes and failure of Jagged-1 expression. We found that TGF-β receptor II signaling regulates Noggin, Wnt9a, and growth and differentiation factor-5 joint morphogenic gene expressions. In Tgfbr2PRX-1KO growth plates adjacent to interphalangeal joints, Indian hedgehog expression is increased, whereas Collagen 10 expression decreased. We propose a model for joint development in which TGF-β signaling represents a means of entry to initiate the process.

Cartilage disorders: potential therapeutic use of mesenchymal stem cells.

Chondrogenesis is a well-orchestrated process driven by chondroprogenitors that undergo to condensation, proliferation and chondrocyte differentiation. Because cartilage lacks regenerative ability, treatments for cartilage diseases are primarily palliative. Adult bone marrow contains a reservoir of mesenchymal stem cells (MSC) with in vitro and in vivo potential of becoming cartilage. To optimize the potential therapeutic use of MSC in cartilage disorders, we need to understand the mechanisms by which growth factors determine their chondrogenic potential. Insulin-like growth factors (IGFs) play a central role in chondrogenesis as indicated by the severe growth failure observed in animals carrying null mutations of Igfs and Igf1R genes. We have found that IGF-I has potent chondrogenic effects in MSC. Effects are similar to transforming growth factor-Beta (TGF-Beta). Insulin-like growth factor binding protein-3 (IGFBP-3), well characterized as IGF carrier, has intrinsic bioactivities that are independent of IGF binding. IGFBP-3 levels are increased in degenerative cartilage diseases such as osteoarthritis. We have demonstrated that IGFBP-3 has IGF-independent growth inhibitory effects in chondroprogenitors. We now show that IGFBP-3 induces MSC apoptosis and antagonizes TGF-Beta chondroinductive effects in chondroprogenitors. Understanding IGF-I chondroinductive and IGFBP-3 chondroinhibitory effects would provide critical information to optimize the therapeutic use of MSC in cartilage disorders.

Subcellular localization of IRS-1 in IGF-I-mediated chondrogenic proliferation, differentiation and hypertrophy of bone marrow mesenchymal stem cells.

Bone marrow derived mesenchymal stem cells (BM-MSC) can differentiate into chondrocytes. Understanding the mechanisms and growth factors that control the MSC stemness is critical to fully implement their therapeutic use in cartilage diseases. The activated type 1 insulin-like growth factor receptor (IGF-IR), interacting with the insulin receptor substrate-1 (IRS-1), can induce cancer cell proliferation and transformation. In cancer or transformed cells, IRS-1 has been shown to localize in the cytoplasm where it activates the canonical Akt pathway, as well as in the nucleus where it binds to nuclear proteins. We have previously demonstrated that IGF-I has distinct time-dependent effect on primary BM-MSC chondrogenic pellets: initially (2-day culture), IGF-I induces proliferation; subsequently, IGF-I promotes chondrocytic differentiation (7-day culture). In the present study, by using MSC from the BM of IRS-1− / − mice we show that IRS-1 mediates almost 50% of the IGF-I mitogenic response and the MAPK-MEK/ERK signalling accounts for the other 50%. After stimulation with IGF-I, we found that in 2-day old human and mouse derived BM-MSC pellets, IRS-1 (total and phosphorylated) is nuclearly localized and that proliferation prevails over differentiation. The IGF-I mitogenic effect is Akt-independent. In 7-day MSC pellets, IGF-I stimulates the chondrogenic differentiation of MSC into chondrocytes, pre-hypertrophic and hypertrophic chondrocytes and IRS-1 accumulates in the cytoplasm. IGF-I-dependent differentiation is exclusively Akt-dependent. Our data indicate that in the physiologically relevant model of primary cultured MSC, IGF-I induces a temporally regulated nuclear or cytoplasmic localization of IRS-1 that correlate with the transition from proliferation to chondrogenic differentiation.

TGF-β Type II Receptor/MCP-5 Axis: At the Crossroad between Joint and Growth Plate Development

Despite its clinical significance, the mechanisms of joint morphogenesis are elusive. By combining laser-capture microdissection for RNA sampling with microarrays, we show that the setting in which joint-forming interzone cells develop is distinct from adjacent growth plate chondrocytes and is characterized by downregulation of chemokines, such as monocyte-chemoattractant protein-5 (MCP-5). Using in vivo, ex vivo, and in vitro approaches, we show that low levels of interzone-MCP-5 are essential for joint formation and contribute to proper growth plate organization. Mice lacking the TGF-β-type-II-receptor (TβRII) in their limbs (Tgfbr2(Prx1KO)), which lack joint development and fail chondrocyte hypertrophy, show upregulation of interzone-MCP-5. In vivo and ex vivo blockade of the sole MCP-5 receptor, CCR2, led to the rescue of joint formation and growth plate maturation in Tgfbr2(Prx1KO) but an acceleration of growth plate mineralization in control mice. Our study characterized the TβRII/MCP-5 axis as an essential crossroad for joint development and endochondral growth.

Goodpasture Antigen-binding Protein and Its Spliced Variant, Ceramide Transfer Protein, Have Different Functions in the Modulation of Apoptosis during Zebrafish Development

Human Goodpasture antigen-binding protein (GPBP) is an atypical protein kinase that phosphorylates the Goodpasture auto-antigen, the α3 chain of collagen IV. The COL4A3BP gene is alternatively spliced producing two protein isoforms: GPBP and GPBPΔ26. The latter lacks a serine-rich domain composed of 26 amino acid residues. Both isoforms also function as ceramide transfer proteins (CERT). Here, we explored the function of Gpbp and GpbpΔ26/CERT during embryogenesis in zebrafish. We cloned both splice variants of the zebrafish gene and found that they are differentially expressed during development. We used antisense oligonucleotide-mediated loss-of-function and synthetic mRNA-based gain-of-function approaches. Our results show that the loss-of-function phenotype is linked to cell death, evident primarily in the muscle of the somites, extensive loss of myelinated tracks, and brain edema. These results indicate that disruption of the nonvesicular ceramide transport is detrimental to normal embryonic development of somites and brain because of increased apoptosis. Moreover, this phenotype is mediated by Gpbp but not GpbpΔ26/CERT, suggesting that Gpbp is an important factor for normal skeletal muscle and brain development.

Loss of the α2β1 integrin alters human papilloma virus-induced squamous carcinoma progression in vivo and in vitro.

Expression of the α2β1 integrin, a receptor for collagens and laminin, is altered during tumor progression. Recent studies have linked polymorphisms in the α2 integrin gene with oral, squamous cell carcinoma (SCC). To determine the α2β1 integrins role in SCC progression, we crossed α2-null mice with K14-HPV16 transgenic animals. Pathological progression to invasive carcinoma was evaluated in HPV-positive, α2-null (HPV/KO) and HPV-positive, wild-type (HPV/WT) animals. α2β1 integrin expression stimulated progression from hyperplasia and papillomatosis to dysplasia with concomitant dermal mast cell infiltration. Moreover, lymph node metastasis was decreased by 31.3% in HPV/KO, compared to HPV/WT, animals. To evaluate the integrin-specific impact on the malignant epithelium versus the microenvironment, we developed primary tumor cell lines. Although transition from dysplasia to carcinoma was unaltered during spontaneous tumor development, isolated primary HPV/KO SCC cell lines demonstrated decreased migration and invasion, compared to HPV/WT cells. When HPV/WT and HPV/KO SCC cells were orthotopically injected into WT or KO hosts, tumor α2β1 integrin expression resulted in decreased tumor latency, regardless of host integrin status. HPV/WT SCC lines failed to demonstrate a proliferative advantage in vitro, however, the HPV/WT tumors demonstrated increased growth compared to HPV/KO SCC lines in vivo. Although contributions of the integrin to the microenvironment cannot be excluded, our studies indicate that α2β1 integrin expression by HPV-transformed keratinocytes modulates SCC growth and progression.

Selective, α2β1 integrin-dependent secretion of il-6 by connective tissue mast cells.

Mast cells, critical mediators of inflammation and anaphylaxis, are poised as one of the first lines of defense against external assault. Mast cells release several classes of preformed and de novo synthesized mediators. Cross-linking of the high-affinity FcΕRI results in degranulation and the release of preformed, proinflammatory mediators including histamine and serotonin. We previously demonstrated that mast cell activation by Listeria monocytogenes requires the α2β1 integrin for rapid IL-6 secretion both in vivo and in vitro. However, the mechanism of IL-6 release is unknown. Here, we demonstrate the Listeria- and α2β1 integrin-mediated mast cell release of preformed IL-6 without the concomitant release of histamine or β-hexosaminidase. α2β1 integrin-dependent mast cell activation and IL-6 release is calcium independent. In contrast, IgE cross-linking-mediated degranulation is calcium dependent and does not result in IL-6 release, demonstrating that distinct stimuli result in the release of specific mediator pools. These studies demonstrate that IL-6 is presynthesized and stored in connective tissue mast cells and can be released from mast cells in response to distinct, α2β1 integrin-dependent stimulation, providing the host with a specific innate immune response without stimulating an allergic reaction.

Abstract 415: Dynamic interplay of tumor and microenvironmental contributions of the α2β1 integrin in squamous cell carcinoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The complex interplay between tumor cells and their surrounding microenvironment define cancer progression. The α2β1 integrin, expressed by subsets of epithelial, endothelial, fibroblast, and inflammatory cells, is a receptor for matrix and non-matrix ligands, including collagens, laminins, and immunomodulatory molecules C1q and collectins. Altered α2β1 integrin expression in cancer has been linked to tumor progression; recent studies have linked polymorphisms in the α2β1 integrin with oral, squamous cell carcinoma (SCC). We hypothesized that the α2β1 integrin functions in SCC initiation or progression by modulating interactions between the squamous epithelium and/or tumor microenvironment. To determine the role of α2β1 integrin expression on SCC progression, we crossed α2β1 integrin-null mice with K14-HPV transgenic animals, a well-established model of epithelial carcinogenesis. Progression from hyperplasia to dysplasia and invasive carcinoma was evaluated in HPV-positive, α2β1 integrin-null (HPV/KO) and HPV-positive, wild-type (HPV/WT) animals. Our data demonstrate that during SCC progression, expression of the α2β1 integrin stimulates progression from hyperplasia and papillomatosis to dysplasia. Decreased dysplasia in HPV/KO animals at early time points of tumor progression directly correlated with reduced dermal mast cell infiltration. Moreover, lymph node metastasis was decreased by 31.3% and associated with alterations in lymphatic vasculature within the tumor microenvironment of HPV/KO, compared to HPV/WT, animals. Since these studies utilized global α2β1 integrin knockout mice, we developed primary tumor cell lines to evaluate the integrin-specific impacts on the malignant epithelium versus the host microenvironment. Although transition from dysplasia to carcinoma was unaltered in our original tumor studies, isolated primary HPV/KO SCC cell lines had decreased invasion in a 3D transwell migration assay, compared to HPV/WT cells. When HPV/WT and HPV/KO SCC cell lines were orthotopically injected into either WT or KO hosts, α2β1 integrin-specific expression on tumor cells decreased tumor latency, regardless of host integrin status. HPV/WT SCC lines failed to demonstrate a proliferative advantage in vitro, however, the HPV/WT tumors demonstrated increased growth rates compared to HPV/KO SCC lines in vivo. Therefore, these latter data suggest that contributions of the tumor microenvironment cannot be excluded and are likely modulating SCC growth characteristics. Our studies indicate the α2β1 integrin plays diverse roles during tumor progression, acting on the tumor and tumor microenvironment. Although the α2β1 integrin plays no obvious role during normal development, exposure to physiological stresses, such as cancer, unmasks functions of the α2β1 integrin to reveal its importance in tissue biology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 415. doi:10.1158/1538-7445.AM2011-415