Maria Trojanowska’s research while affiliated with Boston University and other places

Objective Rarefaction of blood and lymphatic vessels in the skin has been reported in systemic sclerosis (SSc) (scleroderma). E26 transformation–specific–related factor (ERG) and Friend leukemia virus–induced erythroleukemia 1 (FLI‐1) are important regulators of angiogenesis, but their role in lymphatic vasculature is lesser known. The goal of this study was to determine the role of ERG and FLI‐1 in postnatal lymphangiogenesis and SSc lymphatic system defects. Methods Immunofluorescence was used to detect ERG and FLI‐1 in skin biopsy samples from patients with SSc and healthy controls. Transcriptional analysis of ERG or FLI‐1–silenced human dermal lymphatic endothelial cells (LECs) was performed using microarrays. Effects of ERG and FLI‐1 deficiency on in vitro tubulogenesis in human dermal LECs were examined using a Matrigel assay. ERG and FLI‐1 endothelial–specific knockouts and ERG lymphatic–specific knockouts were generated to examine vessel regeneration in mice. Results ERG and FLI‐1 protein levels were reduced in the blood and lymphatic vasculature in SSc skin biopsy samples. ERG levels were shown to regulate genes involved in lymphatic vessel specification, including vascular endothelial growth factor receptor 3/FLT‐4, lymphatic vessel endothelial hyaluronan receptor 1, SOX‐18, and prospero homeobox 1 (PROX‐1), whereas FLI‐1 enhanced the function of ERG. The ERG–FLT‐4 pathway regulated in vitro tubulogenesis in human LECs. Deficiency of ERG or FLI‐1 similarly impaired the function of blood vessels in mice. However, only ERG deficiency affected the regeneration of lymphatic vessels during wound healing. Conclusion ERG and FLI‐1 are essential regulators of blood and lymphatic vessel regeneration. Deficiency of ERG and FLI‐1 in SSc endothelial cells may contribute to the impairment of blood and lymphatic vasculature in patients with SSc. image

Progressive lung fibrosis is associated with poorly understood aging-related endothelial cell dysfunction. To gain insight into endothelial cell alterations in lung fibrosis we performed single cell RNA-sequencing of bleomycin-injured lungs from young and aged mice. Analysis reveals activated cell states enriched for hypoxia, glycolysis and YAP/TAZ activity in ACKR1+ venous and TrkB+ capillary endothelial cells. Endothelial cell activation is prevalent in lungs of aged mice and can also be detected in human fibrotic lungs. Longitudinal single cell RNA-sequencing combined with lineage tracing demonstrate that endothelial activation resolves in young mouse lungs but persists in aged ones, indicating a failure of the aged vasculature to return to quiescence. Genes associated with activated lung endothelial cells states in vivo can be induced in vitro by activating YAP/TAZ. YAP/TAZ also cooperate with BDNF, a TrkB ligand that is reduced in fibrotic lungs, to promote capillary morphogenesis. These findings offer insights into aging-related lung endothelial cell dysfunction that may contribute to defective lung injury repair and persistent fibrosis.

Fibrogenesis is a sequential pathological process culminating in the scarring of virtually any organ. Fibrosis is characterized by disruption of normal tissue architecture and its replacement with stiff, paucicellular, and collagen-rich connective tissue. The process results in progressive functional impairment and ultimately organ failure. Fibrosis is the hallmark of systemic sclerosis (SSc, scleroderma), as well as a large and heterogeneous collection of human fibrotic diseases. In SSc, synchronous fibrosis occurs in the skin and multiple internal organs. Fibrosis is the end result of chronic tissue injury characterized by vascular damage, inflammation, and mesenchymal stromal cell activation in a genetically predisposed individual. Injury-activated vascular, epithelial, and immune cells generate profibrotic soluble mediators that serve as paracrine cues to induce sustained activation, differentiation, and survival of stromal cells, leading to excessive extracellular matrix (ECM) deposition, tissue stiffness, and ultimately fibrosis. Fibrosis progression is driven by multiple self-sustaining amplification mechanisms including mechanical feedback loops, epigenetic modifications, matrikines, metabolic alterations, cellular aging, and senescence. Advances in deciphering the pathophysiology of fibrosis in SSc are paving the way for innovative therapeutic approaches that not only prevent progression of tissue scarring, but also have the potential to reverse established fibrosis. Current approved antifibrotic treatments are modestly slow, but do not reverse fibrosis.

The ETS transcription factor ERG is a master regulator of endothelial gene specificity and highly enriched in the capillary, vein, and arterial endothelial cells. ERG expression is critical for endothelial barrier function, permeability, and vascular inflammation. A dysfunctional vascular endothelial ERG has been shown to impair lung capillary homeostasis, contributing to pulmonary fibrosis as previously observed in IPF lungs. Our preliminary observations indicate that lymphatic endothelial cells (LEC) in the human IPF lung also lack ERG. To understand the role of ERG in pulmonary LECs, we developed LEC-specific inducible Erg-CKO and Erg-GFP-CKO conditional knockout (CKO) mice under Prox1 promoter. Whole lung microarray analysis, flow cytometry, and qPCR confirmed an inflammatory and pro-lymphvasculogenic predisposition in Erg-CKO lung. FITC-Dextran tracing analysis showed an increased pulmonary interstitial lymphatic fluid transport from the lung to the axial lymph node. Single-cell transcriptomics confirmed that genes associated with cell junction integrity were downregulated in Erg-CKO pre-collector and collector LECs. Integrating Single-cell transcriptomics and CellChatDB helped identify LEC specific communication pathways contributing to pulmonary inflammation, trans-endothelial migration, inflammation, and Endo-MT in Erg-CKO lung. Our findings suggest that downregulation of lymphatic Erg crucially affects LEC function, LEC permeability, pulmonary LEC communication pathways and lymphatic transcriptomics.

Pulmonary arterial hypertension (PAH) is a life-threatening condition characterized by a progressive increase in pulmonary vascular resistance leading to right ventricular failure and often death. Here we report that deficiency of transcription factor GATA6 is a shared pathological feature of PA endothelial (PAEC) and smooth muscle cells (PASMC) in human PAH and experimental PH, which is responsible for maintenance of hyper-proliferative cellular phenotypes, pulmonary vascular remodeling and pulmonary hypertension. We further show that GATA6 acts as a transcription factor and direct positive regulator of anti-oxidant enzymes, and its deficiency in PAH/PH pulmonary vascular cells induces oxidative stress and mitochondrial dysfunction. We demonstrate that GATA6 is regulated by the BMP10/BMP receptors axis and its loss in PAECs and PASMC in PAH supports BMPR deficiency. In addition, we have established that GATA6-deficient PAEC, acting in a paracrine manner, increase proliferation and induce other pathological changes in PASMC, supporting the importance of GATA6 in pulmonary vascular cell communication. Treatment with dimethyl fumarate resolved oxidative stress and BMPR deficiency, reversed hemodynamic changes caused by endothelial Gata6 loss in mice, and inhibited proliferation and induced apoptosis in human PAH PASMC, strongly suggesting that targeting GATA6 deficiency may provide a therapeutic advance for patients with PAH.