Jacques E. Nör

Vascular endothelial growth factor (VEGF)-mediated angiogenesis is associated with enhanced endothelial cell survival and induction of Bcl-2 expression.

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen and permeability factor that is potently angiogenic in vivo. We report here studies that suggest that VEGF potentiates angiogenesis in vivo and prolongs the survival of human dermal microvascular endothelial cells (HDMECs) in vitro by inducing expression of the anti-apoptotic protein Bcl-2. Growth-factor-enriched and serum-deficient cultures of HDMECs grown on collagen type I gels with VEGF exhibited a 4-fold and a 1.6-fold reduction, respectively, in the proportion of apoptotic cells. Enhanced HDMEC survival was associated with a dose-dependent increase in Bcl-2 expression and a decrease in the expression of the processed forms of the cysteine protease caspase-3. Cultures of HDMECs transduced with and overexpressing Bcl-2 and deprived of growth factors showed enhanced protection from apoptosis and exhibited a twofold increase in cell number and a fourfold increase in the number of capillary-like sprouts. HDMECs overexpressing Bcl-2 when incorporated into polylactic acid sponges and implanted into SCID mice exhibited a sustained fivefold increase in the number of microvessels and a fourfold decrease in the number of apoptotic cells when examined 7 and 14 days later. These results suggest that the angiogenic activity attributed to VEGF may be due in part to its ability to enhance endothelial cell survival by inducing expression of Bcl-2.

Buonocore Memorial Lecture

For many years, operative dentistry has been using regenerative approaches to treat dental disease. The use of calcium hydroxide to stimulate reparative or reactionary dentin is clearly an example of such a therapeutic strategy. The advent of tissue engineering is allowing dentistry to move forward in the use of regeneration as an underlying principle for the treatment of dental disease. Tissue engineering is a multi-disciplinary science that brings together biology, engineering and clinical sciences with developing new tissues and organs. It is based on fundamental principles that involve the identification of appropriate cells, the development of conducive scaffolds and an understanding of the morphogenic signals required to induce cells to regenerate the tissues that were lost. This review is focused on the presentation and discussion of existing literature that covers the engineering of enamel, dentin and pulp, as well on the engineering of entire teeth. There are clearly major road-blocks to overcome before such strategies move to the clinic and are used regularly to treat patients. However, existing evidence strongly suggests that the engineering of new dental structures to replace tissues lost during the process of caries or trauma will have a place in the future of operative dentistry.Abstract For many years, operative dentistry has been using regenerative approaches to treat dental disease. The use of calcium hydroxide to stimulate reparative or reactionary dentin is clearly an example of such a therapeutic strategy. The advent of tissue engineering is allowing dentistry to move forward in the use of regeneration as an underlying principle for the treatment of dental disease. Tissue engineering is a multi-disciplinary science that brings together biology, engineering and clinical sciences with developing new tissues and organs. It is based on fundamental principles that involve the identification of appropriate cells, the development of conducive scaffolds and an understanding of the morphogenic signals required to induce cells to regenerate the tissues that were lost. This review is focused on the presentation and discussion of existing literature that covers the engineering of enamel, dentin and pulp, as well on the engineering of entire teeth. There are clearly major roadblocks to ...

Dental pulp tissue engineering with stem cells from exfoliated deciduous teeth.

Stem cells from human exfoliated deciduous teeth (SHED) have been isolated and characterized as multipotent cells. However, it is not known whether SHED can generate a dental pulp-like tissue in vivo. The purpose of this study was to evaluate morphologic characteristics of the tissue formed when SHED seeded in biodegradable scaffolds prepared within human tooth slices are transplanted into immunodeficient mice. We observed that the resulting tissue presented architecture and cellularity that closely resemble those of a physiologic dental pulp. Ultrastructural analysis with transmission electron microscopy and immunohistochemistry for dentin sialoprotein suggested that SHED differentiated into odontoblast-like cells in vivo. Notably, SHED also differentiated into endothelial-like cells, as demonstrated by B-galactosidase staining of cells lining the walls of blood-containing vessels in tissues engineered with SHED stably transduced with LacZ. This work suggests that exfoliated deciduous teeth constitute a viable source of stem cells for dental pulp tissue engineering.

Engineering and characterization of functional human microvessels in immunodeficient mice

Current model systems used to investigate angiogenesis in vivo rely on the interpretation of results obtained with nonhuman endothelial cells. Recent advances in tissue engineering and molecular biology suggest the possibility of engineering human microvessels in vivo. Here we show that human dermal microvascular endothelial cells (HDMEC) transplanted into severe combined immunodeficient (SCID) mice on biodegradable polymer matrices differentiate into functional human microvessels that anastomose with the mouse vasculature. HDMEC were stably transduced with Flag epitope or alkaline phosphatase to confirm the human origin of the microvessels. Endothelial cells appeared dispersed throughout the sponge 1 day after transplantation, became organized into empty tubular structures by Day 5, and differentiated into functional microvessels within 7 to 10 days. Human microvessels in SCID mice expressed the physiological markers of angiogenesis: CD31, CD34, vascular cellular adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1). Human endothelial cells became invested by perivascular smooth muscle α-actin–expressing mouse cells 21 days after implantation. This model was used previously to demonstrate that overexpression of the antiapoptotic protein Bcl-2 in HDMEC enhances neovascularization, and that apoptotic disruption of tumor microvessels is associated with apoptosis of surrounding tumor cells. The proposed SCID mouse model of human angiogenesis is ideally suited for the study of the physiology of microvessel development, pathologic neovascular responses such as tumor angiogenesis, and for the development and investigation of strategies designed to enhance the neovascularization of engineered human tissues and organs.

SHED Differentiate into Functional Odontoblasts and Endothelium

Studies on mechanisms underlying the differentiation of dental pulp stem cells are critical for the understanding of the biology of odontogenesis and for dental tissue engineering. Here, we tested the hypothesis that stem cells from exfoliated deciduous teeth (SHED) differentiate into functional odontoblasts and endothelial cells. SHED were seeded in tooth slice/scaffolds and implanted subcutaneously into immunodeficient mice. SHED differentiated into functional odontoblasts that generated tubular dentin, as determined by tetracycline staining and confocal microscopy. These cells also differentiated into vascular endothelial cells, as determined by beta-galactosidase staining of LacZ-tagged SHED. In vitro, vascular endothelial growth factor (VEGF) induced SHED to express VEGFR2, CD31, and VE-Cadherin (markers of endothelium) and to organize into capillary-like sprouts. VEGF induced ERK and AKT phosphorylation (indicative of differentiation), while inhibiting phosphorylation of STAT3 (indicative of ‘stemness’). Collectively, this work demonstrates that SHED can differentiate into angiogenic endothelial cells and odontoblasts capable of generating tubular dentin.

Thrombospondin-1 Induces Endothelial Cell Apoptosis and Inhibits Angiogenesis by Activating the Caspase Death Pathway

Thrombospondin-1 (TSP1) is a potent natural inhibitor of angiogenesis. Although TSP1 has been reported to induce endothelial cell apoptosis in vitro and to downregulate neovascularization in vivo, the molecular mechanisms that link these two processes have yet to be established. Here we report that TSP1 mediates endothelial cell apoptosis and inhibits angiogenesis in association with increased expression of Bax, decreased expression of Bcl-2, and processing of caspase-3 into smaller proapoptotic forms. The ability of TSP1 to induce both endothelial cell apoptosis in vitro and to suppress angiogenesis in vivo was blocked by the caspase-3 inhibitor z-DEVD-FMK. TSP1 also attenuated VEGF-mediated Bcl-2 expression in endothelial cells in vitro and angiogenesis in vivo. Furthermore, TSP1 induced endothelial cell apoptosis and inhibited neovascularization in sponge implants in SCID mice. We conclude that TSP1 induces endothelial cell apoptosis and inhibits neovascularization by altering the profile of survival gene expression and activating caspase-3.

Multiple Roles for the Receptor Tyrosine Kinase Axl in Tumor Formation

A focus of contemporary cancer therapeutic development is the targeting of both the transformed cell and the supporting cellular microenvironment. Cell migration is a fundamental cellular behavior required for the complex interplay between multiple cell types necessary for tumor development. We therefore developed a novel retroviral-based screening technology in primary human endothelial cells to discover genes that control cell migration. We identified the receptor tyrosine kinase Axl as a novel regulator of endothelial cell haptotactic migration towards the matrix factor vitronectin. Using small interfering RNA-mediated silencing and overexpression of wild-type or mutated receptor proteins, we show that Axl is a key regulator of multiple angiogenic behaviors including endothelial cell migration, proliferation, and tube formation in vitro. Moreover, using sustained, retrovirally delivered short hairpin RNA (shRNA) Axl knockdown, we show that Axl is necessary for in vivo angiogenesis in a mouse model. Furthermore, we show that Axl is also required for human breast carcinoma cells to form a tumor in vivo. These findings indicate that Axl regulates processes vital for both neovascularization and tumorigenesis. Disruption of Axl signaling using a small-molecule inhibitor will hence simultaneously affect both the tumor and stromal cell compartments and thus represents a unique approach for cancer therapeutic development.

Endothelial Cell-Initiated Signaling Promotes the Survival and Self-Renewal of Cancer Stem Cells

Recent studies have demonstrated that cancer stem cells play an important role in the pathobiology of head and neck squamous cell carcinomas (HNSCC). However, little is known about functional interactions between head and neck cancer stem-like cells (CSC) and surrounding stromal cells. Here, we used aldehyde dehydrogenase activity and CD44 expression to sort putative stem cells from primary human HNSCC. Implantation of 1,000 CSC (ALDH+CD44+Lin-) led to tumors in 13 (out of 15) mice, whereas 10,000 noncancer stem cells (ALDH-CD44-Lin-) resulted in 2 tumors in 15 mice. These data demonstrated that ALDH and CD44 select a subpopulation of cells that are highly tumorigenic. The ability to self-renew was confirmed by the observation that ALDH+CD44+Lin- cells sorted from human HNSCC formed more spheroids (orospheres) in 3-D agarose matrices or ultra-low attachment plates than controls and were serially passaged in vivo. We observed that approximately 80% of the CSC were located in close proximity (within 100-μm radius) of blood vessels in human tumors, suggesting the existence of perivascular niches in HNSCC. In vitro studies demonstrated that endothelial cell-secreted factors promoted self-renewal of CSC, as demonstrated by the upregulation of Bmi-1 expression and the increase in the number of orospheres as compared with controls. Notably, selective ablation of tumor-associated endothelial cells stably transduced with a caspase-based artificial death switch (iCaspase-9) caused a marked reduction in the fraction of CSC in xenograft tumors. Collectively, these findings indicate that endothelial cell-initiated signaling can enhance the survival and self-renewal of head and neck CSC.

Dentin-derived BMP-2 and Odontoblast Differentiation

It is known that stem cells from exfoliated deciduous teeth (SHED) can be induced to differentiate into odontoblasts. However, the nature of dentin-derived morphogenic signals required for dental pulp stem cell differentiation remains unclear. The hypothesis underlying this work is that dentin-derived Bone Morphogenetic Proteins (BMP) are necessary for the differentiation of SHED into odontoblasts. We observed that SHED express markers of odontoblastic differentiation (DSPP, DMP-1, MEPE) when seeded in human tooth slice/scaffolds and cultured in vitro, or implanted subcutaneously into immunodeficient mice. In contrast, SHED cultured in deproteinized tooth slice/scaffolds, or scaffolds without a tooth slice, do not express these markers. SHED express the BMP receptors BMPR-IA, BMPR-IB, and BMPR-II. Notably, blockade of BMP-2 signaling inhibited the expression of markers of odontoblastic differentiation by SHED cultured in tooth slice/scaffolds. Collectively, this work demonstrates that dentin-derived BMP-2 is required to induce the differentiation of SHED into odontoblasts.

Vascular Endothelial Growth Factor Contributes to the Prostate Cancer-Induced Osteoblast Differentiation Mediated by Bone Morphogenetic Protein

Human prostate cancer has a high predisposition to metastasize to bone, resulting in the formation of osteoblastic metastases. The mechanism through which prostate cancer cells promote osteoblastic lesions is undefined. Vascular endothelial growth factor (VEGF) has been implicated as a mediator of osteoblast activity. In the present study, we examined if prostate cancer cells promote osteoblastic activity through VEGF. We found that LNCaP and C4-2B prostate cancer cell lines and primary tumor and metastatic prostate cancer tissues from patients expressed VEGF. Bone morphogenetic proteins (BMPs), which are normally present in the bone environment, induced VEGF protein and mRNA expression in C4-2B cells. Furthermore, BMP-7 activated the VEGF promoter. Noggin, a BMP inhibitor, diminished VEGF protein expression and promoter activity in C4-2B cells. Conditioned media (CM) from C4-2B cells induced pro-osteoblastic activity (increased alkaline phosphatase, osteocalcin, and mineralization) in osteoblast cells. Both noggin alone and anti-VEGF antibody alone diminished C4-2B CM-induced pro-osteoblastic activity. Transfection of C4-2B cells with VEGF partially rescued the C4-2B CM-induced pro-osteoblastic activity from noggin inhibition. These observations indicate that BMPs promote osteosclerosis through VEGF in prostate cancer metastases. These results suggest a novel function for VEGF in skeletal metastases. Specifically, VEGF promotes osteoblastic lesion formation at prostate cancer bone metastatic sites.