Browsing by Subject "Fibrosis"

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Bronchiolitis Obliterans (BO) is a major cause of chronic airway dysfunction after toxic chemical inhalation. The pathophysiology of BO is not well understood, but epithelial cell injury has been closely associated with the development of fibrotic lesions in human studies and in animal models of both toxin- and transplant-induced BO. However, while almost all cases and models of BO include epithelial injury, not all instances of epithelial injury result in BO, suggesting that epithelial damage per se is not the critical event leading to the development of BO. In this dissertation, we describe a model of chlorine (Cl2)-induced BO in which mice develop tracheal and large airway obliterative lesions within 10 days of exposure to high (350 ppm), but not low (200 ppm), concentrations of Cl2 gas. Lesions develop in a series of well-demarcated pathological changes that include epithelial denudation, inflammatory cell infiltration by day 2 after exposure, fibroblast infiltration and collagen deposition by day 5, and in-growth of blood vessels by day 7, ultimately leading to lethal airway obstruction by days 9-12. Using this model, we were able to test our hypothesis that loss of epithelial progenitor cells is a critical factor leading to the development of obliterative airway lesions after chemical inhalation. Indeed, these lesions arise only under conditions and in areas in which basal cells, the resident progenitor cells for large airway epithelium, are eliminated by Cl2 exposure.

The molecular pathways contributing to BO development are not well understood. Mechanisms of epithelial injury differ across BO models, but we hypothesized that after the inciting injury, BO models share common pathways. We compared microarray analysis from day 5 non-BO- and BO-inducing chemical injuries and subsequently identified biological pathways that may contribute to BO pathogenesis. Our findings add support to pathways previously implicated in BO development and more importantly, suggest potential new pathways and molecular mediators of BO. Furthermore, we evaluated the efficacy of therapeutic inhibition of neovascularization or inflammation to prevent Cl2-induced BO. To date, our therapeutic interventions were ineffective. Nonetheless, our findings suggest that in the context of Cl2-induced BO, vascular endothelial growth factor receptor 2 (a mediator of neovascularization) and inducible nitric oxide synthase (a mediator of inflammation) are not critical in BO pathogenesis.

In sum, our work introduces and characterizes a novel Cl2-induced murine model of BO. Using this model we demonstrated that in the absence of basal cells, epithelial regeneration does not occur and regions of epithelial denudation persist from which an aberrant repair process is initiated, leading to obliterative airway lesions. Our findings suggest that, irrespective of the cause, loss of epithelial progenitor cells may be a critical factor leading to the development of BO. Furthermore, our gene expression analysis implicates novel mediators and signaling pathways in the development of BO. Our analysis lays the foundation for more rigorous exploration of these targets in the pathogenesis of BO.

Muscle fibrosis is caused by muscle injury, dystrophy, sarcopenia, and rheumatoid arthritis. This condition is characterized by hardening and scarring, which impairs contractile muscle function. To understand how fibrotic disease affects muscular function, we created a model of human skeletal muscle fibrosis using three-dimensional engineered skeletal muscle (myobundles). Furthermore, to investigate the effect of skeletal muscle fibrosis on the vascular system, we integrated the fibrotic skeletal muscle with tissue engineered blood vessels. Treating myobundles with Transforming Growth Factor β1 (TGF-β1) reproduced key characteristics of fibrotic skeletal muscle including reduced contractile force, disrupted contractile protein organization, increased stiffness, and expression of profibrotic genes. Treatment with a selective inhibitor (SB525334) of TGF-β1 receptor (ALK5, TGF-βRI) increased contractile function and decreased ECM deposition, consistent with animal studies in the literature. We also observed endogenous secretion of TGF-β1 in our myobundles which is of novel biological significance. siRNA knockdown of TGF-β1 increased contractile force. Testing anti-fibrotic drug Nintedanib in this model, showed an increase in tetanus force production in 2 out of 3 donors and reduction of pro-fibrotic ECM accumulation of collagen 1 and fibronectin. Western blot analysis of Nintedanib also providence evidence of its inhibition of TGF-β1 signaling by the reduction of phosphorylated Smad2/3. Repositioned anti-fibrotic drug Suramin treatment of fibrotic myobundles resulted in increase of tetanus force production in all three donors and reduction of pro-fibrotic ECM accumulation of collagen 1 and fibronectin. Suramin’s influence on TGF-β1 signaling in our system was found not to be as targeted as Nintedanib as there was only reduction in Smad3 phosphorylation and not Smad2 phosphorylation. Anti-fibrotic drug testing in our model was also able to wean out donor specific sensitivity to the drugs with donor 3. Skeletal myobundles were integrated with Tissue Engineered Blood Vessels (TEBVs) to identify the effect of skeletal muscle fibrosis on blood vessels or the human vasculature. Integrated TEBVs with 5 ng/ml TGF-β1 dosed myobundles showed reduced function, increased mesenchymal markers such as vimentin and alpha smooth muscle actin, and increased endothelial cell inflammation. Our results suggest a detrimental effect of skeletal muscle fibrosis on blood vessels and show an interaction between the skeletal muscle fibrosis and the human vasculature This model provides a platform to study skeletal muscle fibrosis alone or its effect on the vasculature and allows for testing anti-fibrotic drugs and assessing myobundle function along with disease influence on human vasculature.

The success of muscle development and regeneration requires cooperation from both myogenic and their supportive niche cells. The muscular niche is complex. At the cellular level it is composed of a broad number of cell types including: endothelial vessels, nerve and nerve-supporting cells, resident immune populations, and a heterogenous group of non-myogenic mesenchymal cells. The non-myogenic mesenchymal cells include pericytes, vascular smooth muscle cells, interstitial tenocyte-like cells, and fibro-adipogenic progenitors (FAPs). Like all members of the muscular niche, this fraction is vital to muscle development and regeneration. Despite their importance to muscle development, regeneration, and homeostasis, detailed identities within non-myogenic mesenchymal cells remain elusive. By understanding the distinct makeup of this population, we can provide a foundation to examine their important regulatory roles in the processes of muscle development, homeostasis, injury and disease.

This thesis utilizes single cell RNA sequencing to establish the populations of non-myogenic mesenchymal cells in developing muscle. Our analysis identified pericytes, vascular smooth muscle cells, and tenocyte-like cell populations while uncovering a new level of heterogeneity in FAPs that not previously appreciated. Despite classical understanding of FAPs as one group, this work found that FAPs were sub-divided into five distinct populations, which compose two trajectories spawning from a common progenitor. This thesis defines the functional differences of each FAP population through a series of experiments including: fluorescence activated cell sorting of various FAP groups, studying their spatial localization on immunofluorescence, and testing the response of different FAPs to multiple injury and disease models.

Separate preliminary work examines the impact of NOTCH signaling in FAPs and the broader non-myogenic mesenchymal cell groups. These studies discovered that NOTCH signaling in the non-myogenic mesenchymal group, but not FAPs specifically, regulates muscular growth and intramuscular adipogenesis. Altogether this thesis advances our understanding of the identity, and role of, non-myogenic mesenchymal cells, in muscle development and regeneration.

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Liver cancer is a devastating disease that is the 5th most common cancer in men, 7th most common cancer in women, and the 3rd leading cause of cancer-related mortality. This disease arises from multiple etiological factors, including hepatitis viruses, environmental toxins, alcohol abuse, and metabolic syndrome, which induce a state of chronic inflammation. This diseased liver tissue background is a drastically different microenvironment from the healthy liver, especially with regards to immune cell prevalence and presence of mediators of immune function. It has been well-established that this altered tissue background contributes significantly to the tumorigenic process, yet its effects on the progression of the disease are more poorly understood.

To better understand the consequences of liver disease on tumor growth and the interplay with its microenvironment, we first utilized two standard methods of fibrosis induction and orthotopic implantation of tumors into the inflamed and fibrotic liver to mimic the liver condition in human HCC patients, and examined the immune infiltrate. Compared to non-diseased controls, tumor growth is significantly enhanced under fibrotic conditions. The immune cells that infiltrated the tumors are also drastically different, with decreased proportions of natural killer cells but greatly increased numbers of immune-suppressive CD11b+ Gr1hi myeloid cells in both models of fibrosis. In addition, there are model-specific differences: increased proportions of CD11b+ myeloid cells and CD4+ CD25+ T-cells are found in tumors in the bile duct ligation model but not in the carbon tetrachloride model. Importantly, the skewed immune infiltration into the tumor, while having some commonalities with the non-tumor tissue, had several distinct, tumor-specific populations. Induction of fibrosis also alters the cytokine production of implanted tumor cells, which could have far-reaching consequences on the immune infiltrate and its functionality. Taken together, this work demonstrates that the combination of fibrosis induction with orthotopic tumor implantation results in a markedly different tumor microenvironment and tumor growth kinetics.

Appreciating that the altered immune microenvironment dramatically shifts tumor progression, we sought to further explore the effects of individual inflammatory mediators on the development of the disease. Interleukin 18 (IL-18) is an inflammatory cytokine that is markedly increased in the circulation of patients with HCC correlated with poor prognosis. However, the precise role for IL-18 in HCC remains unclear, with reports presenting both pro- and anti-tumorigenic activities. To answer this question definitively, we interrogated in more detail the expression profiles of IL-18 in tissue specimens from HCC patients and conducted experimentation using multiple clinically relevant mouse models to explore the functional role of this cytokine in the context of HCC. Our results indicate that IL-18 exerts a tumor-suppressive effect mediated in large part by alterations in survival and functionality of T-lymphocytes which infiltrated the tumor microenvironment. This tumor-suppressive effect is however dependent upon the inflammatory milieu: In the absence of an inflammatory environment, whether from a chemical carcinogenesis model or a fibrosis induction model, loss of IL-18 signaling does not affect tumor growth. This effect is also stage-dependent. Taken together, our findings establish a tumor-suppressive role for IL-18 in established HCC and provide a mechanistic explanation for the complex relationship between its expression pattern and HCC prognosis.

In summary, this work demonstrates a dramatic shift in the microenvironment of developing HCC tumors in the presence of chronic inflammatory stimuli. This microenvironment, which more accurately models the situation in which tumors develop and progress in patients, alters the presence and functionality of many immune mediators. In particular, IL-18 signaling is a powerful mediator of tumor progression, however observation of its functionality is dependent on an inflammatory context. This work provides new insight into the complex processes underlying HCC tumor progression, and emphasizes the necessity for more accurate modeling of HCC progression in mice which takes into account the drastic changes in the tissue caused by chronic liver disease.

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