The vascular endothelium is continuously subjected to a variety of mechanical and chemical stresses while it performs its duties in the maintenance of vascular permeability, tone, hemostasis, inflammation, and remodeling in health and disease. The mechanism by which endothelial cells respond to mechanical forces, or mechanotransduction, is not completely understood and is the subject of ongoing debate. Several theories involving proteins and reactive oxygen species have been proposed as components of the mechanotransduction process, however the role of the lipid microenvironment and lipid signaling remains largely unknown. We hypothesize that mechanical, uniaxial cyclic strain results in an increase in intracellular ceramide in vascular endothelial cells, which participates in signaling necessary to propagate mechanotransduction responses, potentially contributing to early events in the formation of atherosclerotic lesions. To evaluate this hypothesis, we used electrospray mass spectrometry to study the lipid microenvironment, particularly with regards to ceramide signaling, in endothelial cells in response to cyclic strain within and beyond the physiological range, so as to gain a better understanding of the events that may ultimately contribute to endothelial dysfunction. The findings of these studies have elucidated the time scale of the ceramide response and the ceramide biosynthetic and metabolic pathways that occur during the early response to cyclic strain. Ceramide signaling results from distinct signaling events associated with nSMase, aSMase, and de novo ceramide synthesis. The nSMase signaling event appears to be necessary for the later de novo event to occur. The endothelial response to cyclic strain is sensitive to strain magnitude, resulting in ceramide elevation at levels both above and below physiological strain magnitudes, suggestive of a wide variety of arterial pathological states. These findings help to elucidate the early events in the mechanotransduction response to cyclic strain and represent a step towards bridging our understanding of the relationship between mechanotransduction and inflammation as it relates to endothelial cell activation and dysfunction and vascular disease. Establishment of the involvement of the ceramide biosynthetic pathway in endothelial cells and the vascular environment provides us with new biomarkers and therapeutic targets to potentially protect against vascular activation, dysfunction, and atherogenesis.

The complete recovery of injured skeletal muscle has posed a constant challenge for orthopaedic physician. Once injured, skeletal muscle is able to undergo regeneration from satellite cells; nevertheless, in the serious injured muscle, the formation of fibrosis often impedes effective muscle regeneration and resulted in an incomplete muscle healing. Therefore, to develop biological approaches to improve muscle healing, it is crucial to better understand the mechanisms of the skeletal muscle fibrosis. In the current studies, we found that myostatin (MSTN), a member of TGF-â family, plays a role in the formation of skeletal muscle fibrosis, besides the other putative fibrosis stimulator, TGF-â1. In vitro, MSTN directly stimulated the proliferation of fibroblasts and their productions of fibrotic proteins. In vivo, after laceration injury, gastronemius muscles of MSTN-/- mice showed less fibrosis and better muscle regeneration than wide-type (WT) counterparts. Considering MSTN as a therapeutic target of skeletal muscle healing, we found that inhibitors of MSTN, MSTN propeptide (MPRO) and follistatin, effectively blocked MSTN signaling and improved skeletal muscle healing after injured. We used adeno-associated virus (AAV)-mediated MPRO cDNA to successfully deliver MPRO in vivo and improve skeletal muscle healing of normal mice after laceration, and ameliorate dystrophic pathology of mdx/SCID mice. Furthermore, our results demonstrated FLST overexpression (FLST/OE) mice exhibited decreased fibrosis and increased muscle regeneration in injured skeletal muscle as compared to wild-type (WT) mice. Moreover, muscle progenitor cells (MPCs) isolated from MSTN-/- and FLST/OE mice significantly regenerated more myofibers than MPCs obtained from WT mice, when transplanted into dystrophic muscles. Collectively, our results suggested that MSTN directly stimulated fibrosis in the injured skeletal muscle; blocking MSTN signaling with MPRO or FLST improved skeletal muscle healing after laceration injury; blocking MSTN signaling in donor MPCs significantly enhanced the success of cell transplantation into dystrophic muscles. Our studies not only uncovered some of the mechanisms implicated in skeletal muscle fibrosis and regeneration, and help the development of new therapeutic approach for promoting the healing of injured or diseased skeletal muscle, but also render a new sight of how to obtain robust genetically modified cell populations for cell therapy.

BOLD techniques have been used in a vast range of applications including functional MRI (fMRI) and clinical MR venography of brain vasculature. Despite the immense success of BOLD fMRI applications, our understanding of complex neuronal and hemodynamic processes associated with BOLD techniques is limited. An experimental investigation with BOLD MR venography may allow us to expand our knowledge in hemodynamic process involved in BOLD fMRI. BOLD techniques are also clinically useful. In clinical brain imaging studies, imaging both time-of-flight (TOF) MR angiogram (MRA) and BOLD MR venogram (MRV) is often desirable, because they complement the depiction of vascular pathologies. Nevertheless, MRV is usually not acquired to minimize the image acquisition time. It will be highly beneficial if we can acquire MRV while imaging MRA without increasing scan time. Thus, the objective of our study was to develop and assess BOLD MRV techniques for both functional and clinical applications. For the experimental evaluation of BOLD MRV, we used a rat brain model at 9.4T. The scan condition for BOLD MRV was optimized and the venous origin of hypointense vasculature was investigated with modulation of oxygenation. Detailed venules of ˜16-30μm diameter were detected in the resulting in vivo images with 78μm isotropic scan resolution, verified with in vivo two-photon microscopy and computer simulation data. Activation foci of high-resolution BOLD fMRI maps were correlated with relatively large intracortical veins detected with high-resolution BOLD MRV, indicating that detectability of conventional BOLD fMRI is limited by density of these intracortical veins (˜1.5 vessels/mm²). For the clinical application of BOLD MRV, we developed and tested a compatible dual-echo arteriovenography (CODEA) technique for simultaneous acquisition of TOF MRA and BOLD MRV at a 3T human system. Image quality of the CODEA technique acquired in a single session was comparable to conventional TOF MRA and BOLD MRV separately acquired in two sessions. The CODEA technique was applied to chronic stroke studies. Detailed vascular structures including arterial occlusions and venous abnormalities were depicted. The CODEA technique appears valuable to other clinical applications, particularly for those requiring efficient MRA/MRV imaging with limited scan time such as acute stroke studies.

Hallmarks of progression of epithelial-derived tumors include downregulation of cell-cell adhesion, dysregulated cell proliferation, increased resistance to apoptosis and acquisition of motile and invasive phenotype. Profilin-1 (Pfn1) is a ubiquitously expressed actin-binding protein which is required for proliferation and migration of most normal cells, yet its expression is significantly downregulated in various types of adenocarcinoma including those originating in breast, pancreas and liver. Tumor-suppressive action of Pfn1 on breast cancer cells has also been previously documented in the literature. The present study shows that loss of Pfn1 expression in normal human mammary epithelial cells leads to junctional delocalization of E-cadherin with a concomitant reduction in cell-cell adhesion, reduced cell-matrix adhesion, increased cell proliferation and a hypermotile phenotype. These findings may provide a possible insight on why Pfn1 expression is downregulated in breast cancer cells. Using MDA-MB-231 as a model system for mesenchymal breast cancer cell type, we further show that overexpression of Pfn1 can restore adherence junctions and phenotypic reversion to an epithelioid-type through junctional stabilization of an endogenously expressed cadherin molecule. Pfn1 overexpression in MDA-MB-231 cells causes cell-cycle arrest at G1, inhibition in proliferation in vitro and tumor growth in vivo. Pfn1-induced growth inhibition of MDA-MB-231 cells is partly mediated by upregulation of p27kip1 (a CDK inhibitor) in cell-cell adhesion-dependent manner. We finally show that Pfn1 overexpression also sensitizes MDA-MB-231 cells to apoptosis suggesting the survival of breast cancer cells can also be modulated Pfn1. Taken together, these findings highlight for the first time mechanistic insights underlying some of the tumor-suppressive properties of Pfn1.

Premature birth is a major public health problem accounting for over 13,000 deaths and 30,000 surviving infants with life-long morbidity yearly. Preterm premature rupture of the fetal membranes (FM) is the initiating event leading to preterm birth of 40% of these premature infants. The mechanisms which cause FM failure and thereby rupture are not understood. A weak zone in the FM region overlying the cervix has recently been discovered, which demonstrates both biophysical weakness and concomitant biochemical evidence of tissue remodeling and apoptosis. This weak zone has been the subject of limited biomechanical investigation. A full understanding of FM failure requires a complete characterization of structural and biomechanical behavior of the FM at near/full term under sub-failure (forces well below that which induce rupture) and failure conditions as well as elucidating the biological factors which modulate its failure. The goals of this study were to (1) characterize the sub-failure structure-strength behavior of the FM under physiological loading conditions, (2) implement a structural constitutive model to investigate the sub-failure responses of FM, and (3) characterize the structural/strength sequelae during failure of the FM in the non-weak regions. The first two aims of this study established the baseline structure-strength relation of non-weak zone FM tissue. Specifically, the stress-strain relationship from an unloaded state up to failure was established. It was found that the FM behaves as an effectively isotropic material and that collagen fibers of the FM are recruited rapidly once they are loaded, which may be an important mechanism in the facilitation of FM rupture. Finally, a novel membrane inflation device revealed that upon loading, there was a small increase in collagen fiber alignment, although not significant, and there was no change in collagen fiber direction. More importantly, FM failure occurs catastrophically, suggesting that particular collagen fibers are not predisposed to failure. The results obtained in this study further our understanding of this unique physiological event, failure, and provide a basis for establishing how the structure-function relationship of the FM is altered the weak zone, which has unique mechanical properties that facilitates membrane rupture.

Recent advances in neural prosthetics have provided patients with the ability to use signals derived from motor areas of the cerebral cortex to directly control an external device under visually guided closed-loop control. To attain a more natural form of prosthesis control, it is desirable to develop systems capable of providing real-time somatosensory feedback as well as visual feedback, akin to how we naturally process sensory information to control our limbs. To this end, a sophisticated data acquisition, control and feedback system was developed for neural prosthetics and psychophysics research. The system deterministically collects and processes high volume neural ensemble activity, limb kinematics, and eye movements while generating visual stimuli in an immersive three-dimensional virtual reality (VR) environment. A vibrotactile feedback device was also developed and incorporated into the system. It delivers real-time limb kinematics feedback in the form of continuous, graded vibratory stimulation. A flexible and intuitive user interface allows the researcher to design experimental paradigms and adjust parameters on the fly during experiments. A psychophysical study was conducted using this system to evaluate the potential use of vibrotactile feedback as a sensory substitution method to provide somatosensory feedback for neural prosthesis control. The study also aimed to provide insight into the mechanisms of multimodal sensory processing and sensory-motor control. Able-bodied human subjects performed a trajectory-following reach task in the VR environment under different visual and vibrotactile feedback conditions. The study showed that vibrotactile feedback is capable of enhancing motor performance, implying that subjects were able to integrate and effectively use this new 'proprioceptive-like' sensory modality. Subjects were also able to partially maintain task performance using vibrotactile feedback in the absence of visual feedback. Improved motor learning and motor skill consolidation were also observed after training in the VR environment with vibrotactile feedback. These results suggest that vibrotactile feedback may be a viable method for delivering somatosensory feedback for applications such as neural prosthesis control, motor rehabilitation, and enhanced human-computer interaction.

Smooth muscle cell (SMC) embedded scaffolds have possible applications in treating diseased tissues that are rich in SMCs. Stress urinary incontinence (SUI) is an example of a disease that can be caused due to SMC dysfunction within the urinary sphincter. The goal of this thesis was to create a SMC-populated tissue engineered urethral wrap (TEUW) using autologous urethral SMCs (uSMCs), to be used as a cuff around the native urethra to integrate with the host tissue for providing mechanical and functional reinforcement to the diseased urethra. uSMCs were isolated from rat urethras. SMC phenotype was verified by immunofluorescence and western blotting. Isolation purity was assessed by staining uSMCs for skeletal muscle and urothelium markers since they are also present in the urethra. TEUWs were examined for SMC phenotype, apoptosis, mechanical and histological endpoints after culture. This thesis also evaluated the functionality of differentiated SMCs (dSMCs), which were derived via mechanical stimulation of bone marrow-derived mesenchymal stem cell (BMMSCs). The long-term objective is to use BMMSCs as an autologous source for SMCs in order to create TEUW-like tubular constructs for treating SMC related dysfunctions including, but not limited to SUI. uSMCs and dSMCs were assessed and compared for intracellular Ca2+ activity (fura-2) and contractile responses (live-cell) to various stimuli. Results of isolated uSMCs revealed expression of SMC markers and absence of skeletal and urothelium markers, suggesting isolation purity. uSMC-based TEUWs showed non-linear pressure-diameter profiles like soft tissues, greater compliance than the native urethra, and burst pressures similar to stem-cell based TEUWs. Both, uSMCs and dSMCs, exhibited intracellular Ca2+ activity, with and without extracellular Ca2+, vital for full SMC function. However, their failure to show morphological changes in the presence of agonists during contractility assessment indicated absence of mature SMCs. In summary, this study demonstrates proficient uSMC isolation, which represents an important step towards TEUW development, and that uSMCs and dSMCs are not fully functional at the differentiation stage tested. Future work should focus on increasing contractile protein expression by using matrix-like culture systems and/or biochemical stimulants. Following a systematic examination, SMC-populated TEUWs could be tested in an animal model.

This project was aimed at further elucidating the role of protein kinase C (PKC)-induced phosphorylation of troponin-I (cTnI) in cardiac contraction. We created a new transgenic mouse model (TG-E), expressing a mutant cardiac TnI constitutively pseudo-phosphorylated at the three PKC phosphorylation sites (S43, S45, T144 mutated to glutamate). 2D-DIGE (Difference in Gel Electrophoresis) gels indicated 7.2 ± 0.5% replacement, with no change in baseline level of actual phosphorylation of cTnI or other myofilamental proteins. Experiments were conducted in perfused isolated mouse hearts, isolated papillary muscles, and skinned fiber preparations. The mechanical measurements were complemented by biochemical and molecular biological measurements, and a mathematical model-based analysis for integrative interpretation. Compared to wild-type mice, TG-E mice exhibited negative inotropy in in vivo echocardiographic studies (9% decrease in fractional shortening), isolated hearts (14% decrease in peak developed pressure), papillary muscles (53% decrease in maximum developed force), and skinned fibers (14% decrease in maximally activated force, Fmax). Additionally, TG-E mice exhibited slowed relaxation in echocardiographic studies, isolated hearts and intact papillary muscles. The TG-E mice showed no differences in calcium sensitivity, cooperativity, steady-state force-ATPase relationship, and calcium transient (amplitude and relaxation). The four-state model of cardiac contraction was used for a model-based analysis of the data. The model was verified as a priori globally identifiable using a differential algebraic approach. The model-based analysis revealed that experimental observations in TG-E mice could be reproduced by two simultaneous perturbations: a decrease in the rate of crossbridge formation and an increase in calcium-independent persistence of the myofilament active state. In summary, a modest increase in PKC-induced cTnI phosphorylation can significantly regulate cardiac muscle contraction: (1) negative inotropy via decreased crossbridge formation and (2) negative lusitropy via persistence of myofilament active state. Based on our data and data from the literature we speculate that the effects of PKC-mediated cTnI phosphorylation are site-specific (S43/S45 vs. T144).

Biologic scaffolds composed of extracellular matrix (ECM) have been successfully used as templates for the constructive remodeling of numerous tissues in preclinical studies and human clinical applications. The mechanisms by which ECM induces remodeling are largely unknown, but the degradation products of ECM may play key roles in constructive remodeling. This dissertation investigated the hypothesis that ECM degradation products possess chemoattractant properties for progenitor cell (PC) populations that participate in constructive remodeling. We investigated different methods of in vitro degradation of ECM and determined physiologically relevant methods of degradation yielded degradation products with chemoattractant activity. Both pepsin and collagenase digestion of ECM resulted in chemoattraction of two distinct PC populations. We then investigated if ECM degradation products from a given tissue have more potent chemoattractant properties for PCs derived from the same tissue type than for PCs derived from other tissues. Although ECM derived from skin, liver, small intestine, and urinary bladder were all chemoattractive for at least one PC population, a tissue-specific chemotactic effect was not observed in studies using skin, liver, and intestinal PCs. However, results showed that the age and species from which ECM is harvested has an effect on the chemoattractant potential of the ECM for some PC populations. We investigated if prevention or retardation of ECM degradation in vivo would reduce bone marrow-derived PC involvement in constructive remodeling, yielding a different histomorphologic outcome than normal ECM degradation. Bone marrow-derived cells (BMCs) participated in the early remodeling of wounded mouse skin treated with rapidly degrading ECM scaffolds. By 28 days post-surgery, the number of BMCs returned to normal levels, suggesting that these cells do not participate in long-term constructive remodeling of mouse skin. Slowly degrading chemically crosslinked ECM scaffolds did not recruit more BMCs than are found in normal uninjured mouse skin at any time investigated. Wounds treated with rapidly degrading ECM appeared to remodel more rapidly than other treatment groups. These results suggest that scaffold type affects the temporal remodeling of injured mouse skin and that while BMCs participate in remodeling of skin wounds in mice, local tissue cells may also play an important role.

Approximately 5-8% of Americans are affected by some type of autoimmune disease. The current standard of care for these patients involves pharmacologic therapy to induce systemic immunosuppression, which carries significant side effects. There is a need for therapies that effectively restore immune-mediated tolerance towards the body, but do not cause inappropriate immunosupression. The following dissertation details the synthesis, characterization, and performance of tolerogenic dendritic cells (DC), as well as the exosomes that they secrete. Exosomes are small (40-100 nm) membrane-bound microvesicles produced by reverse budding of the membrane of the multivesicular endosome in DC, as well as many other cell types. DC were transduced with adenoviral vectors expressing target genes in order to manipulate their function. After transduction, DC and their exosomes were examined for the ability to modulate disease-induced inflammation in the NOD mouse model of type 1 diabetes and the delayed-type hypersensitivity (DTH) model. DC transduced with an IL-4 expressing vector are capable or preventing the onset of hyperglycemia when administered to 12-weeks-old and 12-16 week-old prediabetic mice. Treated mice demonstrate modulation of T-cell mediated β-cell autoimmunity, including reduced insulitis, increased FoxP3 expression in the islet-draining lymph nodes, and Th2 skewing of islet-antigen induced cytokine secretion profile. We further demonstrate that transduction of DC with adenoviral vectors expressing indoleamine 2,3-diooxygenase (IDO) and CTLA-4-Ig are efficient means to generate IDO+ DC in vitro, and that these DC effectively reduce paw swelling in the DTH model of antigen-specific inflammation. The exosomes secreted by these IDO+ DC contain IDO protein, and suppress DTH responses at a level comparable to their parental DC in a manner dependent on B7-1 and B7-2. To confirm the role of B7 costimulatory molecules on tolerogenic DC and exosomes, we further demonstrate that B7-1 and B7-2, but not PD-L1 or PD-L2, are required for in vivo suppressive activity of rIL-10 treated DC and their secreted exosomes. This endeavor has increased the knowledge regarding therapeutic DC and exosomes. We have demonstrated strategies to generate tolerogenic DC and exosomes, as well as factors that are required for their activity in vivo.