Project Summary Age-dependent impairment of lung regeneration and repair contributes to the pathogenesis of aging- associated chronic lung diseases (CLD). Angiogenesis – the formation of new blood capillaries- plays a key role in lung regeneration and is impaired in aging animals. Thus, in order to develop more efficient therapies for aging-associated CLD, we need to understand the mechanisms by which aging impairs angiogenesis in the lung. In addition to soluble growth factors and signaling molecules, biophysical factors alter angiogenic gene expression and regulate angiogenesis. We have reported that changes in cell size and geometry control endothelial cell (EC) growth. It is known that cell size increases during aging in various tissues. However, the effects of age-dependent increases in EC size on impairment of angiogenesis in the aged lung and the underlying mechanism have not been explored. The overall goal of this proposal is to characterize the mechanosensitive mechanism of age-dependent decline in angiogenesis, and to leverage this knowledge to develop an efficient strategy for CLD. Angiopoietin (Ang)-Tie2 signaling controls angiogenesis and plays important roles in lung development and regeneration. The mechanosensitive transcriptional co-activator, Yes- associated protein (YAP1), senses various mechanical forces and controls organ development and regeneration. YAP1 stimulates angiogenesis through angiopoietin2 (Ang2) signaling and deregulation of YAP1 is involved in the pathogenesis of aging-associated CLD. Our new preliminary data demonstrate that pulmonary ECs in aged mice are significantly larger than those in young mice. The levels of YAP1 are lower and EC proliferation is inhibited in ECs isolated from aged mouse lungs compared to those from younger mouse lungs. YAP1 knockdown decreases Ang2 expression in mouse lung ECs and the levels of Ang2 is lower in aged mouse lung ECs. When we culture aged lung ECs on single-cell sized fibronectin-coated large islands comparable in size to aged mouse lung EC, YAP1 is excluded from the nucleus and is inactive, while reduction of aged EC size restores YAP1 nuclear localization. Vascular network formation is inhibited in the fibrin gel implanted on the aged mouse lungs. We hypothesize that age-dependent increases in EC size may suppress aged EC proliferation and angiogenesis in the aged mouse lung through aberrant YAP1-Ang2 signaling. In Aim1, we will investigate the effects of age-dependent changes in EC size on EC proliferation, apoptosis, and senescence using the microcontact printing system. In Aim2, we will determine whether EC size-dependent changes in YAP1 signaling mediates impairment of angiogenesis in the aged lung through Ang2. Our idea to focus on the effects of age-related changes in EC size on angiogenesis is highly unique and innovative advances. If this study proves that reduction of aged EC size reverses the age-dependent decline in vascular morphogenesis in the aged lungs thr...