Browsing by Subject "Senescence"

Clinical studies have identified strong correlations between aging and the development of atherosclerosis. In particular, endothelial cell senescence is implicated in age-related changes in vasoreactivity. Oxidative stress is considered the primary source of endothelial cell (EC) senescence in vivo. EC senescence leads to abnormal proliferation of vascular smooth muscle cells, reduced vasoreactivity, enhanced vascular permeability, and greater adhesion of circulating monocytes and lipids. Endothelial senescence often occurs coincident with an inflammatory response within the endothelium. Recapitulating this mechanism of inducing EC senescence in vitro will facilitate a more precise understanding of how aging contributes to endothelial dysfunction and development of vascular diseases, particularly atherosclerosis. Additionally, evidence of vascular remodeling, particularly deposition of fibronectin and stiffening of the vessel wall matrix, is found in both older patients and atheroprone regions. The independent effects of these factors on the function of endothelial cells is poorly understood due to the inability to study them in isolation in vivo. The Truskey lab developed tissue-engineered blood vessels (TEBVs) which recapitulate the structure and function of an arteriole in vitro. These vessels can be fabricated rapidly, perfused immediately after fabrication, and reach functional maturity after a week. Measurements of endothelium-mediated vascular function confirm the presence of a healthy endothelium in the vessels for several weeks after initial fabrication. This in vitro system allows more precise control over the cellular and structural components of blood vessels than is possible with in vivo experiments. Ultimately, the development of a more robust in vitro model for atherosclerosis will contribute to an increased understanding of vascular disease progression and provide a platform for the evaluation of new drugs during preclinical trials. Specific Aim 1: Evaluate the functional effects of stress-induced senescence on TEBVs. Stress-induced senescence reduced endothelium-dependent vessel function and resulted in endothelial cell inflammation with minimal effects on the surrounding hNDFs. Stress-induced senescence was induced in vitro by treatment with hydrogen peroxide. 2-D cells and TEBVs were treated for 5 or 7 days with hydrogen peroxide. Cells in 2-D were stained for p21 to evaluate senescence, as well as key immune cell adhesion markers VCAM-1, ICAM-1, and E-Selectin. To characterize the effects on TEBVs, vasoreactivity in response to an endothelium-independent vasodilator (sodium nitroprusside) and vasoconstrictor (phenylephrine) were quantified, as well as endothelium-dependent vasoreactivity (acetylcholine). Immunostaining of p21 and VCAM-1 expression was also used to confirm that senescence and inflammation were induced in the TEBVs alongside the reduction in endothelium-dependent vasodilation. Specific Aim 2: Evaluate the capacity of stress-induced senescence to increase the monocyte adhesion and foam cell formation in the TEBVs Stress-induced senescence in TEBVs increased adhesion of circulating monocytes and foam cell formation in accumulated monocytes and medial hNDFs. Senescence was induced as in Aim 1, by treating vessels with hydrogen peroxide. The resulting increase in senescence, VCAM-1, and E-selectin increased adhesion of circulating monocytes to the vessel wall. To develop an atherogenesis model, low density lipoprotein was enzymatically modified into a more inflammatory state which is often identified within atherosclerotic lesions. Introducing enzyme-modified low-density lipoprotein (eLDL) alongside hydrogen peroxide treatment further increased endothelial cell activation. There was a significant increase in the percentage of ICAM-1 positive cells when eLDL was applied to endothelial cells alongside H2O2. hNDFs absorbed and retained eLDL, even without H2O2 in the growth media. When TEBVs were exposed to a combination of eLDL, H2O2, and cell-tracker red monocytes, endothelium-dependent vasoreactivity was significantly compromised. Lipid retention within the vessel wall was significant, as was adhesion of monocytes. Specific Aim 3: Evaluate the drug-responsiveness of the TEBV senescence model and the ability of geroprotective agents to reduce senescence-induced vascular dysfunction, monocyte adhesion, and foam cell formation. Development of a physiologically relevant model for vascular senescence can provide a valuable tool for evaluating the efficacy of drugs targeting atherosclerosis, particularly a new class of drugs in development called senolytics. Senolytics, and their sister drugs senomorphics, specifically target senescent cells and transiently disable the anti-apoptotic pathways that prolong their lives, reducing the burden of senescent cells within the tissue. Senomorphics target factors within the senescence-associated secretory pathway (SASP) to reduce cytokine production and inflammation. Dasatinib and quercetin, two senolytics, and tacrolimus, a senomorphic, were tested on CBECFCs growing in 2-D to see if they were effective at reducing the percentage of p21 positive (senescent) cells. Tacrolimus was found to be the best candidate and used in TEBV trials. TEBVs treated with tacrolimus for 48 hours after induction of senescence recovered significantly more endothelium-dependent vasoreactivity compared to vessels left to recover from H2O2 in normal growth media. Additionally, addition of tacrolimus for the duration of hydrogen peroxide treatment had an atheroprotective effect. Adhesion of monocytes and foam cell formation were significantly reduced compared to vessels without tacrolimus. In summary, the work presented here demonstrates that a TEBV model of vascular senescence can be generated in under two weeks using near-physiological levels of hydrogen peroxide. This model can be capitalized upon to model atherogenesis by adding only eLDL and monocytes. We were also able to effectively use the senomorphic tacrolimus to mitigate the effects of senescence on monocyte adhesion and lipid uptake. This system could be used to investigate other senolytics or test the efficacy and toxicity of novel drugs still in development.

The eukaryotic epigenome has an instrumental role in determining and maintaining cell identity and function. Epigenetic components such as DNA methylation, histone tail modifications, chromatin accessibility, and DNA architecture are tightly correlated to central cellular processes, while their dysregulation manifests in aberrant gene expression and disease. The ability to specifically edit the epigenome holds the promise of enhancing understanding how epigenetic modifications function and enabling manipulation of cell phenotype for scientific or therapeutic purposes. Genome targeting technologies, such as the CRISPR/Cas9 system, have successfully been harnessed to create epigenome editing tools to alter gene expression. Prominently, two leading CRISPR-based technologies, CRISPRa and CRISPRi, were shown to be highly specific and effective in controlling gene transcription levels. These tools, however, often lead to formation of complexes that affect a multitude of endogenous factors, thus mitigating our ability to elucidate the role of individual epigenetic marks. Moreover, changes in epigenetic marks are associated with numerous health conditions, therefore the development of tools that can modify specific marks may help in creating disease models, or the restoration of a “healthy” epigenome. We first created a suite of CRISPR-based epigenome modifiers (CRISPR-GEMs) that were aimed to catalyze the removal or addition of specific histone tail marks. Next, we tested a few promising CRISPR-GEMs on multiple target genes to characterize their effect on gene expression and chromatin marks. Furthermore, we utilized these tools to deepen our insights into the relationship of individual histone marks and gene expression in different contexts and to better our understanding of the kinetics and dynamics of several of these novel tools alongside existing ones. Additionally, we decided to use the CRISPRa platform to explore senescence, a cellular process that is at the epicenter of aging and has been shown to play a key role in various age-related diseases. Using the CRISPRa platform in an inducible-senescence cell model, we found and validated multiple transcription factors (TFs) that regulate senescence-associated growth arrest (SAGA). Lastly, we characterized genetic pathways that are pivotal to successful inhibition of SAGA, thereby demonstrating a new application of epigenome editing in a senescence model that enhanced our understanding of the pathways that govern SAGA.

Epstein-Barr virus (EBV) is an oncogenic gamma-herpesvirus that infects over 90% of adults worldwide. Typically, EBV establishes a benign latent infection that is controlled by a strong cytotoxic T cell immune response. However, EBV infection in immunocompromised patients has been associated with development of several lymphoid and epithelial cell malignancies, including Burkitt’s lymphoma, Hodgkin’s lymphoma, post-transplant lymphoproliferative disease, and nasopharyngeal carcinoma. In primary human B cells, EBV infection has been shown to induce a transient period of hyper-proliferation, but many of these infected cells succumb to a DNA damage response (DDR)-mediated growth arrest. We hypothesize that EBV infection triggers replicative stress early after infection and facilitates persistent activation of the DDR establishing oncogene-induced senescence. To test this hypothesis, we infected primary human B cells with EBV and examined cellular proliferation and host DNA damage response pathways at early and late stages post infection. We found that early after EBV infection, rapidly proliferating B cells exhibited signs of replication stress and reduced levels of purine dNTP nucleotide pools that are necessary for sustained proliferation. These findings suggest that purine dNTP biosynthesis plays a critical role in the early stages of EBV-mediated B cell immortalization. Furthermore, we observed the formation of persistent DDR foci in arrested B cells and identified key regulators of long-term outgrowth of EBV-infected B cells. Ultimately, this work has shown that early after EBV infection, cells that experience aberrant proliferation establish oncogene-induced senescence by chronically activating the DDR in the context of reduced dNTP nucleotide pools.

Abstract

The trefoil factor family of secreted proteins contains three members; trefoil factor 1 or TFF1, trefoil factor 2 or TFF2, and trefoil factor 3 or TFF3. These three proteins share a conserved 42-43 amino acid domain containing 6 cysteine residues resulting in three disulfide bonds that holds the protein in a characteristic three-loop or "trefoil structure" known as the P domain. TFF1 is primarily localized to the stomach and secreted by the gastric mucosa while TFF2 and TFF3 are primarily localized to the colon and duodenum and secreted by the goblet cells. All three of these proteins play a protective role in the gastrointestinal tract where they are normally localized and have been identified as possible tumor suppressors, however, these proteins are also upregulated in cancer within tissues where they are not normally expressed including the breast, pancreas, prostate, and liver. The mechanisms by which two of these factors, TFF1 and TFF3, promote tumorigenesis remain largely undefined. In this dissertation we will attempt to elucidate these mechanisms as well as the regulation of these two proteins in both pancreatic and prostate cancer. Many of the underlying genetic and molecular mechanisms involved in the development of both pancreatic and prostate cancer remain largely unknown and as a result, therapeutic and diagnostic tools for treating these diseases are not as effective as they could be. By deciphering the role of TFF1 and TFF3 in these cancers, they could potentially serve as new therapeutic targets or biomarkers for treating both diseases.

Chapter 2 of this dissertation will examine the functional role of TFF1 promoting tumorigenesis in pancreatic and prostate cancer. We will show that TFF1 expression is critical for the viability of both pancreatic and prostate cancer cells and that reduction of TFF1 expression in these cells results in decreased tumorigenicity when implanted in immunocompromised mice. It will also be demonstrated that TFF1's function in promoting tumorigenicity is its ability to assist tumor cells overcome the tumor suppressive barrier of senescence. Thirdly, we show that the form of senescence that TFF1 assists in allowing the cells overcome is oncogene-induced senescence (OIS). Lastly, a cell cycle array identifies the potential downstream target p21CIP, a cyclin-dependent kinase inhibitor and OIS marker, whose expression is induced by loss of TFF1 expression.

In Chapter 3 of this work, we examine the role of another trefoil factor family member, TFF3, and its role in promoting prostate tumorigenesis. Just as with TFF1, it appears that TFF3 3 expression is critical for prostate cancer cell viability and tumorigenicity using the same experimental techniques used in Chapter 2. Using a genetically defined model of prostate cancer, a PI3-kinase-dependent regulatory mechanism of TFF3 emerges in this prostate cancer context. Using this system we begin to see a divergence in both regulation and function of TFF1 and TFF3 in prostate cancer. Finally, a mouse model expressing TFF3 was developed to monitor the histopthological changes associated with expression of this protein. Initial characterization of this model suggests a hyperplastic phenotype coinciding with TFF3 expression in the prostate.

The two studies in this dissertation establish a role of TFF1 and TFF3 in both prostate and pancreatic tumorigenesis and demonstrate that ablation of expression of both proteins is a potent inhibitor of tumorigenesis. With this knowledge, it is possible that TFF1 and TFF3 may become a potential therapeutic target or diagnostic marker for better treatment of prostate and pancreatic cancer.

Cellular senescence is a unique cell fate characterized by stable cell cycle arrest and the extensive production and secretion of various cytokines, chemokines, proteases, and growth factors, a phenomenon known as the senescence-associated secretory phenotype (SASP). Although secreted factors are known to have important biological effects on both senescent and non-senescent cells in the contexts of normal aging and disease, the precise molecular mechanisms responsible for generating a SASP in response to senescent stimuli have remained largely obscure. To identify the major initiator, we used an unbiased profiling strategy and discovered a multi-ligand scavenger receptor CD36 is rapidly upregulated in multiple cell types in response to replicative, oncogenic and chemical senescent stimuli. Moreover, ectopic CD36 expression in dividing mammalian cells is sufficient to initiate the production of a large subset of known components of the SASP via activation of the canonical Src-NFκB pathway, resulting in the subsequent onset of a full senescent state. The CD36-mediated secretome is further shown to be ligand-dependent, as fibroblast cultures lacking the CD36 ligand amyloid beta (Aβ) are unresponsive to CD36 upregulation but can be driven to senesce by the addition of exogenous ligand. Finally, loss-of-function experiments revealed a strict requirement for CD36 in secretory molecule production during conventional senescence reprogramming. These results uncover the Aβ-CD36-NFκB signaling axis as an important regulator of the senescent cell fate via induction of the SASP.

To further explore the possible implication of Aβ-CD36-NFκB-SASP signaling, we found that the CD36 expression is significantly down-regulated in the context of lung malignant tissues, specifically in cancer cells. Subsequent explorations revealed CD36 as a strong tumor suppressor by secreting pro-inflammatory cytokines and recruiting cytotoxic T. For the CD36 ligand - Aβ, we observed a major accumulation in the tumor region which might serve as the tumor-suppressing signaling initiation cue once CD36 is introduced. The findings indicate a possible tumor suppressive signaling lead by Aβ-CD36.

Taken together, we discovered a novel signaling of Aβ-CD36-NFκB in regulating SASP during the process of lung aging and the progression of lung malignancy.