Vascular remodeling in patients with rare genetic disorders

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Monogenetic disorders grant the unique ability to understand complex diseases due to a single defined genetic defect. Advanced sequencing technology accelerates the discovery of gene candidates but progress can be slowed by the inability to distinguish between functional disease related and non-disease related mutations. We use patient-specific in vitro disease modeling systems combined with genetic tools to identify disease related mutation, to study gene related disease mechanism and to perform drug screening. Further, comprehensive clinical evaluation of patients with these conditions further help elucidate the disease mechanism and the organ systems affected. Arterial calcifications contribute to morbidity and are predictors of premature death. Vascular calcification is a secondary complication to diseases such as atherosclerosis, diabetes mellitus type II and chronic kidney disease. Pathomechanism is poorly understood. We identified a novel genetic disease, ACDC, in which de novo vascular calcifications form in the lower extremity arteries of affected adults in a consanguineous family (n = 5) as well as several unrelated individuals (n = 3). All individuals have mutations in the NT5E gene encoding CD73, an enzyme involved in the extracellular purine metabolic pathway. CD73, the affected enzyme, normally converts extracellular AMP to adenosine and inorganic phosphate. Cultured fibroblasts isolated from affected individuals show complete loss of CD73 enzyme activity and these cells display increased tissue non-specific alkaline phosphatase (TNAP) activity, a key enzyme for calcification. Genetic rescue and treatment with adenosine reduced both TNAP activity and in vitro calcification in the patient cells, suggesting that adenosine signaling in normal tissue inhibits ectopic vessel calcification. Adenosine signaling has been shown to be protective during harmful events in the cardiovascular system, however the link between adenosine and protection from calcification is unprecedented. Further, four of our ACDC patients underwent a joint biopsy procedure to remove joint calcification. Sample analysis allowed us to identify calcium pyrophosphate and hydroxyapatite crystals in these samples by microscopy and micro tomography, respectively. Autosomal dominant hyper-IgE syndrome (AD-HIES; Jobs syndrome) is a rare immunodeficiency affecting fewer than 1 per million people worldwide. In 2007, mutations in the STAT3 gene were identified as the genetic cause of AD-HIES. In addition to multiple life-threatening infections, such as recurrent staphylococcal abscesses and pyogenic pneumonias starting early in childhood, the disease is accompanied by multiple abnormalities outside the immune system. As opposed to complete tissue healing experienced by otherwise healthy patients after pneumonia, aberrant healing often occurs in AD-HIES patients. Vascular abnormalities include arterial tortuosity and abnormal dilatation and aneurysms of medium sized arteries, all of which can lead to potentially fatal complications such as myocardial infarction and subarachnoid hemorrhage. Despite an identified genetic cause, the exact mechanisms of the non-hematopoietic AD-HIES pathologies are not well understood. Our analysis of healing of small skin wounds created by removing cylindrical piece of skin (2mm) revealed delayed granulation tissue formation and vascularization in AD-HIES patients. Using RNA-Seq analysis of skin fibroblasts isolated from these patients, we identified deficiencies in angiogenesis, extracellular matrix metabolism, and wound healing signaling pathways mediated by dysregulation of HIF1 signaling. HIF1 expression was reduced in AD-HIES skin wounds. We functionally verified these AD-HIES deficiencies in cell culture and mouse models of angiogenesis: cell culture vessel tube formation assay, teratoma growth assay, a mouse hind-limb ischemia and skin wound healing models. We also revealed abnormal organization of the extracellular matrix and decreased HIF1 expression in coronary arteries of AD-HIES patients linking vascular structural abnormalities to the aberrant STAT3-HIF1a signaling. Several autoinflammatory diseases, such as SAVI, DADA2, NOMID and CANDLE, negatively impact the health of blood vessels and can result in severe tissue damage as well as fatal outcomes for the subjects affected. These diseases are often caused by genetic mutations but the underlying mechanisms have not been well characterized. We are investigating the genetic characteristics of these diseases through clinical evaluation, genetic testing to include whole genome sequencing, high content screening and single cell sequencing as well as patient derived cells (myeloid) to investigate the disease mechanisms in vitro and in vivo murine models. In particular, SAVI patients present with early-onset systemic inflammation, cutaneous vasculopathy, and life-threaten complication pulmonary fibrosis. We have previously found overactivation of STING resulted in chronic activation of the type 1 interferon pathway and endothelial dysfunction. However, the mechanism of pulmonary fibrosis in SAVI patients was largely unknown. Endothelial-to-mesenchymal transition (EndMT) is a major mechanism contributing to multiple organ fibrosis. During the reporting period, we identified perivascular fibrosis with some endothelial and mesenchymal double positive stained cells in SAVI patient lung sections. We also found that EndMT spontaneously developed in induced pluripotent stem cell-derived endothelial cells (iECs) from patients but not in control iECs by downregulating EC markers (VE-cadherin, CD31) and activating ACTA2 (vascular smooth muscle actin) and a mesenchymal differentiation program. Correction of the STING mutation using CRISPR-Cas9-mediated gene editing abrogated EndMT of iECs from one SAVI patient. These data suggest TMEM173/STING gain-of-function mutations lead to EndMT in SAVI patients. Our findings suggest STING overactivation-mediated EndMT through JAK/STAT activation could be a principal mechanism in causing lung fibrosis in SAVI patients. Small vessel diseases are conditions with narrowing of small arteries leading to an imbalance of blood supply upon demand, resulting in progressive chronic hypoperfusion with detrimental outcomes. CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarct and leukoencephalopathy) is caused by mutations in NOTCH3 and is of slow onset (initial clinical manifestations in the third and fourth decade of life) but progressive and fatal with no available cure. Predominant clinical features include migraines, strokes, memory loss, and multiple psychiatric symptoms including dementia. Similarly, Degos disease is a poorly understood rare genetic disorder leading to defects in coagulation, in which small veins and arteries become occluded. Initial symptoms present as skin lesions for both the benign and systemic forms of the disease. However, benign Degos is limited to skin lesions, while systemic Degos progresses to other organ systems (intestines, CNS) and becomes fatal within 2-3 years of diagnosis. We are continuing to work on characterizing the etiology and natural history of CADASIL and Degos patients through comprehensive clinical and molecular evaluations as well as to develop patient-derived disease models to better understand the pathophysiology of these conditions.