Role of Vinculin in Regulating Force-Sensitive Dynamics of Adherens Junctions During Collective Cell Migration

Dynamic processes such as morphogenesis and tissue patterning require the precise control of many cellular processes, especially cell migration. Historically, these processes are thought to be mediated by genetic and biochemical signaling pathways. However, recent advances have unraveled a previously unappreciated role of mechanical forces in regulating these homeostatic processes in of multicellular systems. In multicellular systems cells adhere to both deformable extracellular matrix (ECM) and other cells, which are sources of applied forces and means of mechanical support. Cells detect and respond to these mechanical signals through a poorly understood process called mechanotransduction, which can have profound effects on processes such as cell migration. These effects are largely mediated by the sub cellular structures that link cells to the ECM, called focal adhesions (FAs), or cells to other cells, termed adherens junctions (AJs).

Overall this thesis is comprised of my work on identifying a novel force dependent function of vinculin, a protein which resides in both FAs and AJs - in dynamic process of collective migration. Using a collective migration assay as a model for collective cell behavior and a fluorescence resonance energy transfer (FRET) based molecular tension sensor for vinculin I demonstrated a spatial gradient of tension across vinculin in the direction of migration. To define this novel force-dependent role of vinculin in collective migration I took advantage of previously established shRNA based vinculin knock down Marin-Darby Canine Kidney (MDCK) epithelial cells.

The first part of my thesis comprises of my work demonstrating the mechanosensitive role of vinculin at AJ’s in collectively migrating cells. Using vinculin knockdown cells and vinculin mutants, which specifically disrupt vinculin’s ability to bind actin (VinI997A) or disrupt its ability to localize to AJs without affecting its localization at FAs (VinY822F), I establish a role of force across vinculin in E-cadherin internalization and clipping. Furthermore by measuring E-cadherin dynamics using fluorescence recovery after bleaching (FRAP) analysis I show that vinculin inhibition affects the turnover of E-cadherin at AJs. Together these data reveal a novel mechanosensitive role of vinculin in E-cadherin internalization and turnover in a migrating cell layer, which is contrary to the previously identified role of vinculin in potentiating E-cadherin junctions in a static monolayer.

For the last part of my thesis I designed a novel tension sensor to probe tension across N-cadherin (NTS). N-cadherin plays a critical role in cardiomyocytes, vascular smooth muscle cells, neurons and neural crest cells. Similar to E-cadherin, N-cadherin is also believed to bear tension and play a role in mechanotransduction pathways. To identify the role of tension across N-cadherin I designed a novel FRET-based molecular tension sensor for N-cadherin. I tested the ability of NTS to sense molecular tension in vascular smooth muscle cells, cardiomyocytes and cancer cells. Finally in collaboration with the Horwitz lab we have been able to show a role of tension across N-cadherin in synaptogenesis of neurons.

c-Myc is required for maintenance of glioma cancer stem cells.

BACKGROUND: Malignant gliomas rank among the most lethal cancers. Gliomas display a striking cellular heterogeneity with a hierarchy of differentiation states. Recent studies support the existence of cancer stem cells in gliomas that are functionally defined by their capacity for extensive self-renewal and formation of secondary tumors that phenocopy the original tumors. As the c-Myc oncoprotein has recognized roles in normal stem cell biology, we hypothesized that c-Myc may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells. METHODOLOGY/PRINCIPAL FINDINGS: Based on previous methods that we and others have employed, tumor cell populations were enriched or depleted for cancer stem cells using the stem cell marker CD133 (Prominin-1). We characterized c-Myc expression in matched tumor cell populations using real time PCR, immunoblotting, immunofluorescence and flow cytometry. Here we report that c-Myc is highly expressed in glioma cancer stem cells relative to non-stem glioma cells. To interrogate the significance of c-Myc expression in glioma cancer stem cells, we targeted its expression using lentivirally transduced short hairpin RNA (shRNA). Knockdown of c-Myc in glioma cancer stem cells reduced proliferation with concomitant cell cycle arrest in the G(0)/G(1) phase and increased apoptosis. Non-stem glioma cells displayed limited dependence on c-Myc expression for survival and proliferation. Further, glioma cancer stem cells with decreased c-Myc levels failed to form neurospheres in vitro or tumors when xenotransplanted into the brains of immunocompromised mice. CONCLUSIONS/SIGNIFICANCE: These findings support a central role of c-Myc in regulating proliferation and survival of glioma cancer stem cells. Targeting core stem cell pathways may offer improved therapeutic approaches for advanced cancers.

Diet-induced obesity alters the differentiation potential of stem cells isolated from bone marrow, adipose tissue and infrapatellar fat pad: the effects of free fatty acids.

INTRODUCTION: Obesity is a major risk factor for several musculoskeletal conditions that are characterized by an imbalance of tissue remodeling. Adult stem cells are closely associated with the remodeling and potential repair of several mesodermally derived tissues such as fat, bone and cartilage. We hypothesized that obesity would alter the frequency, proliferation, multipotency and immunophenotype of adult stem cells from a variety of tissues. MATERIALS AND METHODS: Bone marrow-derived mesenchymal stem cells (MSCs), subcutaneous adipose-derived stem cells (sqASCs) and infrapatellar fat pad-derived stem cells (IFP cells) were isolated from lean and high-fat diet-induced obese mice, and their cellular properties were examined. To test the hypothesis that changes in stem cell properties were due to the increased systemic levels of free fatty acids (FFAs), we further investigated the effects of FFAs on lean stem cells in vitro. RESULTS: Obese mice showed a trend toward increased prevalence of MSCs and sqASCs in the stromal tissues. While no significant differences in cell proliferation were observed in vitro, the differentiation potential of all types of stem cells was altered by obesity. MSCs from obese mice demonstrated decreased adipogenic, osteogenic and chondrogenic potential. Obese sqASCs and IFP cells showed increased adipogenic and osteogenic differentiation, but decreased chondrogenic ability. Obese MSCs also showed decreased CD105 and increased platelet-derived growth factor receptor α expression, consistent with decreased chondrogenic potential. FFA treatment of lean stem cells significantly altered their multipotency but did not completely recapitulate the properties of obese stem cells. CONCLUSIONS: These findings support the hypothesis that obesity alters the properties of adult stem cells in a manner that depends on the cell source. These effects may be regulated in part by increased levels of FFAs, but may involve other obesity-associated cytokines. These findings contribute to our understanding of mesenchymal tissue remodeling with obesity, as well as the development of autologous stem cell therapies for obese patients.

Differentiation of mouse induced pluripotent stem cells (iPSCs) into nucleus pulposus-like cells in vitro.

A large percentage of the population may be expected to experience painful symptoms or disability associated with intervertebral disc (IVD) degeneration - a condition characterized by diminished integrity of tissue components. Great interest exists in the use of autologous or allogeneic cells delivered to the degenerated IVD to promote matrix regeneration. Induced pluripotent stem cells (iPSCs), derived from a patient's own somatic cells, have demonstrated their capacity to differentiate into various cell types although their potential to differentiate into an IVD cell has not yet been demonstrated. The overall objective of this study was to assess the possibility of generating iPSC-derived nucleus pulposus (NP) cells in a mouse model, a cell population that is entirely derived from notochord. This study employed magnetic activated cell sorting (MACS) to isolate a CD24(+) iPSC subpopulation. Notochordal cell-related gene expression was analyzed in this CD24(+) cell fraction via real time RT-PCR. CD24(+) iPSCs were then cultured in a laminin-rich culture system for up to 28 days, and the mouse NP phenotype was assessed by immunostaining. This study also focused on producing a more conducive environment for NP differentiation of mouse iPSCs with addition of low oxygen tension and notochordal cell conditioned medium (NCCM) to the culture platform. iPSCs were evaluated for an ability to adopt an NP-like phenotype through a combination of immunostaining and biochemical assays. Results demonstrated that a CD24(+) fraction of mouse iPSCs could be retrieved and differentiated into a population that could synthesize matrix components similar to that in native NP. Likewise, the addition of a hypoxic environment and NCCM induced a similar phenotypic result. In conclusion, this study suggests that mouse iPSCs have the potential to differentiate into NP-like cells and suggests the possibility that they may be used as a novel cell source for cellular therapy in the IVD.

An active learning approach for rapid characterization of endothelial cells in human tumors.

Currently, no available pathological or molecular measures of tumor angiogenesis predict response to antiangiogenic therapies used in clinical practice. Recognizing that tumor endothelial cells (EC) and EC activation and survival signaling are the direct targets of these therapies, we sought to develop an automated platform for quantifying activity of critical signaling pathways and other biological events in EC of patient tumors by histopathology. Computer image analysis of EC in highly heterogeneous human tumors by a statistical classifier trained using examples selected by human experts performed poorly due to subjectivity and selection bias. We hypothesized that the analysis can be optimized by a more active process to aid experts in identifying informative training examples. To test this hypothesis, we incorporated a novel active learning (AL) algorithm into FARSIGHT image analysis software that aids the expert by seeking out informative examples for the operator to label. The resulting FARSIGHT-AL system identified EC with specificity and sensitivity consistently greater than 0.9 and outperformed traditional supervised classification algorithms. The system modeled individual operator preferences and generated reproducible results. Using the results of EC classification, we also quantified proliferation (Ki67) and activity in important signal transduction pathways (MAP kinase, STAT3) in immunostained human clear cell renal cell carcinoma and other tumors. FARSIGHT-AL enables characterization of EC in conventionally preserved human tumors in a more automated process suitable for testing and validating in clinical trials. The results of our study support a unique opportunity for quantifying angiogenesis in a manner that can now be tested for its ability to identify novel predictive and response biomarkers.

B cells in rheumatoid synovitis.

In rheumatoid arthritis, T cells, B cells, macrophages, and dendritic cells invade the synovial membranes, establishing complex microstructures that promote inflammatory/tissue destructive lesions. B cell involvement has been considered to be limited to autoantibody production. However, recent studies suggest that B cells support rheumatoid disease through other mechanisms. A critical element of rheumatoid synovitis is the process of ectopic lymphoid neogenesis, with highly efficient lymphoid architectures established in a nonlymphoid tissue site. Rheumatoid synovitis recapitulates the pathways of lymph node formation, and B cells play a key role in this process. Furthermore, studies of rheumatoid lesions implanted in immunodeficient mice suggest that T cell activation in synovitis is B cell dependent, indicating the role played by B cells in presenting antigens and providing survival signals.

A Tissue-Engineered Microvascular System to Evaluate Vascular Progenitor Cells for Angiogenic Therapies

The ability of tissue engineered constructs to replace diseased or damaged organs is limited without the incorporation of a functional vascular system. To design microvasculature that recapitulates the vascular niche functions for each tissue in the body, we investigated the following hypotheses: (1) cocultures of human umbilical cord blood-derived endothelial progenitor cells (hCB-EPCs) with mural cells can produce the microenvironmental cues necessary to support physiological microvessel formation in vitro; (2) poly(ethylene glycol) (PEG) hydrogel systems can support 3D microvessel formation by hCB-EPCs in coculture with mural cells; (3) mesenchymal cells, derived from either umbilical cord blood (MPCs) or bone marrow (MSCs), can serve as mural cells upon coculture with hCB-EPCs. Coculture ratios between 0.2 (16,000 cells/cm2) and 0.6 (48,000 cells/cm2) of hCB-EPCs plated upon 3.3 µg/ml of fibronectin-coated tissue culture plastic with (80,000 cells/cm2) of human aortic smooth muscle cells (SMCs), results in robust microvessel structures observable for several weeks in vitro. Endothelial basal media (EBM-2, Lonza) with 9% v/v fetal bovine serum (FBS) could support viability of both hCB-EPCs and SMCs. Coculture spatial arrangement of hCB-EPCs and SMCs significantly affected network formation with mixed systems showing greater connectivity and increased solution levels of angiogenic cytokines than lamellar systems. We extended this model into a 3D system by encapsulation of a 1 to 1 ratio of hCB-EPC and SMCs (30,000 cells/µl) within hydrogels of PEG-conjugated RGDS adhesive peptide (3.5 mM) and PEG-conjugated protease sensitive peptide (6 mM). Robust hCB-EPC microvessels formed within the gel with invasion up to 150 µm depths and parameters of total tubule length (12 mm/mm2), branch points (127/mm2), and average tubule thickness (27 µm). 3D hCB-EPC microvessels showed quiescence of hCB-EPCs (<1% proliferating cells), lumen formation, expression of EC proteins connexin 32 and VE-cadherin, eNOS, basement membrane formation by collagen IV and laminin, and perivascular investment of PDGFR-β+/α-SMA+ cells. MPCs present in <15% of isolations displayed >98% expression for mural markers PDGFR-β, α-SMA, NG2 and supported hCB-EPC by day 14 of coculture with total tubule lengths near 12 mm/mm2. hCB-EPCs cocultured with MSCs underwent cell loss by day 10 with a 4-fold reduction in CD31/PECAM+ cells, in comparison to controls of hCB-EPCs in SMC coculture. Changing the coculture media to endothelial growth media (EBM-2 + 2% v/v FBS + EGM-2 supplement containing VEGF, FGF-2, EGF, hydrocortisone, IGF-1, ascorbic acid, and heparin), promoted stable hCB-EPC network formation in MSC cocultures over 2 weeks in vitro, with total segment length per image area of 9 mm/mm2. Taken together, these findings demonstrate a tissue engineered system that can be utilized to evaluate vascular progenitor cells for angiogenic therapies.

CD45+ Cells Present Within Mesenchymal Stem Cell Populations Affect Network Formation of Blood-Derived Endothelial Outgrowth Cells.

Mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) represent promising cell sources for angiogenic therapies. There are, however, conflicting reports regarding the ability of MSCs to support network formation of endothelial cells. The goal of this study was to assess the ability of human bone marrow-derived MSCs to support network formation of endothelial outgrowth cells (EOCs) derived from umbilical cord blood EPCs. We hypothesized that upon in vitro coculture, MSCs and EOCs promote a microenvironment conducive for EOC network formation without the addition of angiogenic growth supplements. EOC networks formed by coculture with MSCs underwent regression and cell loss by day 10 with a near 4-fold and 2-fold reduction in branch points and mean segment length, respectively, in comparison with networks formed by coculture vascular smooth muscle cell (SMC) cocultures. EOC network regression in MSC cocultures was not caused by lack of vascular endothelial growth factor (VEGF)-A or changes in TGF-β1 or Ang-2 supernatant concentrations in comparison with SMC cocultures. Removal of CD45+ cells from MSCs improved EOC network formation through a 2-fold increase in total segment length and number of branch points in comparison to unsorted MSCs by day 6. These improvements, however, were not sustained by day 10. CD45 expression in MSC cocultures correlated with EOC network regression with a 5-fold increase between day 6 and day 10 of culture. The addition of supplemental growth factors VEGF, fibroblastic growth factor-2, EGF, hydrocortisone, insulin growth factor-1, ascorbic acid, and heparin to MSC cocultures promoted stable EOC network formation over 2 weeks in vitro, without affecting CD45 expression, as evidenced by a lack of significant differences in total segment length (p=0.96). These findings demonstrate the ability of MSCs to support EOC network formation correlates with removal of CD45+ cells and improves upon the addition of soluble growth factors.