Identifying Mesenchymal Pathways That Initiate Emphysema in Order to Develop Targeted Therapies.

RATIONALE: Chronic obstructive pulmonary disease (COPD) is the third leading cause of mortality in the United States. COPD and associated emphysematous loss of tissue structure often is exacerbated by vasculopathy, which substantially worsens prognosis and limits survival. Vasculopathy is characterized by remodeling and loss of microvessels. Evidence has also highlighted a role for the alterations to the microvasculature during the early pathogenesis and heterogeneity of disease, although the underlying mechanisms are not defined.

OBJECTIVES: The goal of this proposal is to address this knowledge gap by defining the molecular mechanisms whereby reciprocal mesenchymal progenitor-endothelial cell interactions regulate pulmonary microvascular structure and the development of emphysema. It is generally assumed that vasculopathies associated with emphysema/COPD are attributable to deficient vascular endothelium or smooth muscle cell phenotypes. We challenge this paradigm by defining an adult mesenchymal progenitor cell (ATP-binding cassette transporter [ABCG2]pos MPC) that regulates both microvascular function during tissue homeostasis and adaptive angiogenesis in response to injury. These studies were designed to define the molecular mechanisms whereby reciprocal mesenchymal progenitor-endothelial cell interactions regulate pulmonary microvascular structure and the development of emphysema. We hypothesize that enhanced Wnt/β-catenin signaling within MPCs indirectly leads to emphysema by altering normal MPC-microvascular endothelial cell (MVEC) interactions.

METHODS: To evaluate the role of MPC and β-catenin signaling in lung microvascular homeostasis, we stabilized β-catenin by engineering a conditional activator [β-catenin lacking degradation sites; CatnbloxP(ex3) ], targeted to lung MPCs using ABCG2CreERT2 with reporters: Rosa26m-tdTomato/mGFPlox-stop . This lineage-labeling mouse model, in the presence or absence of Wnt activation, was subjected to vascular injury using SUGEN 5416 and hypoxia. Physiological endpoints of vascular injury; morphometrics, including stereology to measure mean linear intercept (MLI); and lineage analysis were performed. Murine and human MPCs, normal and COPD, were isolated by flow cytometry and analyzed in vitro in coculture analysis with MVECs to define physiological relevance of the cell-cell interactions.

RESULTS: Using a combination of flow cytometry and a low-dose tamoxifen induction lineage-tracing strategy, we defined and validated ABCG2 as a marker to reproducibly label a subpopulation of adult mesenchymal lung progenitor cells (ABCG2pos MPCs) in murine and human lungs. To define a function of lung MPCs during tissue pulmonary homeostasis we perturbed β-catenin signaling. In response to systemic vascular endothelial growth factor inhibition, enhanced Wnt/β-catenin signaling in MPCs drives the appearance of an exacerbated emphysema-like airspace enlargement, with increased vasculopathy with decreased microvessel density, and increased MLI in lung. Interestingly, vascular endothelial growth factor inhibition alone and Wnt activation alone decreased microvessel density without measureable changes in MLI, suggesting that this subtle change may precede loss of tissue structure. Lineage analysis demonstrated that only the Wnt-activated MPC lungs developed abnormal vascularization by the MPC lineage, forming structures lacking traditional smooth muscle or endothelium but resembling microvessels in the distal lung. In vitro, we demonstrate that the Wnt-activated murine MPCs express a similar gene profile to human COPD MPCs, which also exhibit enhanced Wnt signaling. Coculture studies of MPCs with MVECs and analysis of barrier function demonstrated that human COPD MPCs negatively affect barrier function.

CONCLUSIONS: We demonstrate, for the first time, that deregulation of lung MPC function during the development of emphysema drives vasculopathy, which regulates loss of distal lung tissue structure. Interestingly, with the Wnt-activated MPCs exacerbating the loss of microvessel density, there is parallel de novo angiogenesis by the MPCs. MPCs are therefore a previously unstudied compartment of the lung that modulates MVECs and microvascular function, and that demands further study to identify novel therapeutic pathways relevant to the vascular pathobiology of emphysema. The long-term goal of these studies is to promote microvascular function and attenuate emphysema.