Pulmonary hypertension (PH) is a devastating pulmonary vascular disease characterized by aberrant muscularization of the normally nonmuscularized distal pulmonary arterioles. The expression of the transcription factor, Twist1, increases in the lungs of patients with pulmonary arterial hypertension. However, the mechanisms by which Twist1 controls the pathogenesis of PH remain unclear. It is becoming clear that endothelial-to-mesenchymal transition (EndMT) contributes to various vascular pathologies, including PH; Twist1 is known to mediate EndMT. In this report, we demonstrate that Twist1 overexpression increases transforming growth factor (TGF) β receptor2 (TGF-βR2) expression and Smad2 phosphorylation, and induces EndMT in cultured human pulmonary arterial endothelial (HPAE) cells, whereas a mutant construct of Twist1 at the serine 42 residue (Twist1S42A) fails to induce EndMT. We also implanted fibrin gel supplemented with HPAE cells on the mouse lung, and found that these HPAE cells form vascular structures and that Twist1-overexpressing HPAE cells undergo EndMT in the gel, whereas Twist1S42A-overexpressing cells do not. Furthermore, hypoxia-induced EndMT is inhibited in endothelial cells overexpressing Twist1S42A mutant construct in vitro. Hypoxia-induced accumulation of α-smooth muscle actin-positive cells in the pulmonary arterioles is attenuated in Tie2-specific Twist1 conditional knockout mice in vivo. These findings suggest that Twist1 serine 42 phosphorylation plays a key role in EndMT through TGF-β signaling and that modulation of Twist1 phosphorylation could be an effective strategy for managing PH.

Angiogenesis, the formation of new blood capillaries, plays a key role in organ development and regeneration. Inhibition of lung angiogenesis through the blockade of angiogenic signaling pathways impairs compensatory and regenerative lung growth after unilateral pneumonectomy (PNX). The Hippo signaling transducer, Yes-associated protein (YAP1) binds to TEA domain transcription factor (TEAD) and controls organ size and regeneration. However, the role of endothelial YAP1 in lung vascular and alveolar morphogenesis remains unclear. In this report, we have demonstrated that knockdown of YAP1 in endothelial cells (ECs) decreases angiogenic factor receptor Tie2 expression, and inhibits EC sprouting and epithelial cell budding in vitro and vascular and alveolar morphogenesis in the gel implanted on the mouse lung. The expression levels of YAP1, TEAD1, and Tie2 increase in ECs isolated from the remaining mouse lungs after unilateral PNX and vascular formation is stimulated in the post-PNX mouse lungs. Knockdown of endothelial YAP1 inhibits compensatory lung growth and vascular and alveolar morphogenesis after unilateral PNX. These findings suggest that endothelial YAP1 is required for lung vascular and alveolar regeneration and modulation of YAP1 in ECs may be novel interventions for the improvement of lung regeneration.

Mitochondria contribute to key processes of cellular function, while mitochondrial dysfunction is implicated in metabolic disorders, neurodegenerative diseases, and cardiovascular diseases, in which angiogenesis - the formation of new blood capillaries - is dysregulated. The Hippo signaling transducer, Yes-associated protein (YAP1) binds to the TEA domain (TEAD1) transcription factor and controls angiogenesis. YAP1 also regulates glucose metabolism through peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC1α), a major player controlling mitochondrial biogenesis. However, the role of YAP1-TEAD1-PGC1α signaling in mitochondrial structure, cellular metabolism, and angiogenesis in endothelial cells (ECs) remains unclear. We now find that knockdown of TEAD1 decreases the expression of PGC1α and suppresses mitochondrial biogenesis, glycolysis, and oxygen consumption in ECs. A YAP1 mutant construct, YAP1S127A, which stimulates binding of YAP1 to TEAD1, upregulates the expression of PGC1α, induces mitochondrial biogenesis, and increases oxygen consumption and glycolytic flux in ECs; in contrast, YAP1S94A, which fails to bind to TEAD1, attenuates these effects. PGC1α knockdown inhibits YAP1S127A-induced EC sprouting in vitro and vascular morphogenesis in the fibrin gel subcutaneously implanted on mice, while overexpression of PGC1α reverses vascular morphogenesis suppressed by YAP1S94A. These results suggest that YAP1-TEAD1 signaling induces mitochondrial biogenesis in ECs and stimulates angiogenesis through PGC1α. Modulation of YAP1-TEAD1-PGC1α signaling in ECs may provide a novel intervention for angiogenesis-related diseases.

Remodeling of distal pulmonary arterioles (pAs) associated with marked accumulation of pulmonary artery smooth muscle cells (pASMcs) represents one of the major pathologic features of pulmonary hypertension (PH). We have reported that the transcription factor Twist1 mediates hypoxia-induced PH. However, the mechanism by which endothelial Twist1 stimulates SMC accumulation to distal PAs in PH remains unclear. Here, we have demonstrated that Twist1 overexpression increases the expression of platelet-derived growth factor (PDGFB) in human pulmonary arterial endothelial (HPAE) cells. Hypoxia upregulates the levels of Twist1 and PDGFB in HPAE cells. When we implant hydrogel supplemented with endothelial cells (ECs) on the mouse lung, these ECs form vascular lumen structures and hypoxia upregulates PDGFB expression and stimulates accumulation of αSMA-positive cells in the gel, while knockdown of endothelial Twist1 suppresses the effects. The levels of Twist1 and PDGFB are higher in pAe cells isolated from idiopathic pulmonary arterial hypertension (ipAH) patients compared to those from healthy controls. IPAH patient-derived PAE cells stimulate accumulation of αSMA-positive cells in the implanted gel, while Twist1 knockdown in PAE cells inhibits the effects. Endothelial Twist1-PDGFB signaling plays a key role in αSMA-positive cell proliferation and migration in PH. Pulmonary hypertension (PH) is a multifactorial life-threatening cardiopulmonary disorder 1,2. It is characterized by a sustained increase in pulmonary arterial (PA) pressure, leading to right-sided heart failure 1,2. The current therapeutic options only partially improve symptoms and survival 1,3. Uncontrolled pulmonary artery smooth muscle cell (PASMC) proliferation and accumulation of PASMCs to normally non-muscularized distal PAs are the major histological characteristics of PH 2,4. Thus, we need to understand the mechanism of abnormal SMC proliferation, migration, and their accumulation to distal PAs. In addition to lining vascular structures, endothelial cells (ECs) regulate various physiological functions by secreting angiocrine factors 5. Deregulation of this paracrine mechanism leads to the development of diseases 5. In PH pathology, abnormal ECs release factors that stimulate SMC proliferation (e.g., FGF-2 6 , serotonin 7) or fail to produce factors that physiologically suppress SMC proliferation (e.g., apelin 8), indicating that aberrant pulmonary arterial EC signaling plays key roles in abnormal SMC accumulation to distal PAs 2. The transcription factor Twist1 controls physiological and pathological angiogenesis in various organs including the lungs 9-14. The levels of Twist1 are upregulated in the lungs of patients with pulmonary arterial hypertension (PAH; WHO group1 PH) 15,16 and mice with type II bone morphogenetic protein receptor (Bmpr2) gene mutation 15 , a gene mutated in familial and idiopathic PAH 17. Twist1 expression also increases in the patients with chronic lung diseases associated with PH such as pulmonary fibrosis 18,19. We have d...

Angiogenesis – the growth of new blood capillaries- is impaired in aging animals. Biophysical factors such as changes in cell size control endothelial cell (EC) proliferation and differentiation. However, the effects of aging on EC size and the mechanism by which changes in cell size control age-dependent decline in EC proliferation are largely unknown. Here, we have demonstrated that aged ECs are larger than young ECs and that age-dependent increases in EC size control EC proliferation and senescence through CDC42-Yes-associated protein (YAP1) signaling. Reduction of aged EC size by culturing on single-cell sized fibronectin-coated smaller islands decreases CDC42 activity, stimulates YAP1 nuclear translocation and attenuates EC senescence. Stimulation of YAP1 or inhibition of CDC42 activity in aged ECs also restores blood vessel formation. Age-dependent changes in EC size and/or CDC42 and YAP1 activity may be the key control point of age-related decline in angiogenesis.

Aging is associated with impaired angiogenesis and lung alveolar regeneration, which contributes to the increased susceptibility to chronic lung diseases (CLD). We have reported that the Wnt ligand co-receptor, low-density lipoprotein receptor-related protein 5 (LRP5), stimulates angiogenesis and lung alveolar regeneration. However, the role of LRP5 in age-related decline in vascular and alveolar morphogenesis remains unclear. In this report, we have demonstrated that vascular and alveolar structures are disrupted in the 24-month (24M) old mouse lungs. The expression of LRP5 and the major angiogenic factors, VEGFR2 and Tie2, is lower in endothelial cells (ECs) isolated from 24M old mouse lungs compared to those from 2M old mouse lungs. Vascular and alveolar formation is attenuated in the hydrogel implanted on the 24M old mouse lungs, while overexpression of LRP5, which restores angiogenic factor expression, reverses vascular and alveolar morphogenesis in the gel. Compensatory lung growth after unilateral pneumonectomy is inhibited in 24M old mice, which is reversed by overexpression of LRP5. These results suggest that LRP5 mediates age-related inhibition of angiogenesis and alveolar morphogenesis. Modulation of LRP5 may be a novel intervention to rejuvenate regenerative ability in aged lung and will lead to the development of efficient strategies for aging-associated CLD.