Because adult lung tissue has limited regeneration capacity, lung transplantation is the primary therapy for severely damaged lungs. To explore whether lung tissue can be regenerated in vitro, we treated lungs from adult rats using a procedure that removes cellular components but leaves behind a scaffold of extracellular matrix that retains the hierarchical branching structures of airways and vasculature. We then used a bioreactor to culture pulmonary epithelium and vascular endothelium on the acellular lung matrix. The seeded epithelium displayed remarkable hierarchical organization within the matrix, and the seeded endothelial cells efficiently repopulated the vascular compartment. In vitro, the mechanical characteristics of the engineered lungs were similar to those of native lung tissue, and when implanted into rats in vivo for short time intervals (45 to 120 minutes) the engineered lungs participated in gas exchange. Although representing only an initial step toward the ultimate goal of generating fully functional lungs in vitro, these results suggest that repopulation of lung matrix is a viable strategy for lung regeneration.

VEGF is the key growth factor regulating arterial morphogenesis. However, molecular events involved in this process have not been elucidated. Synectin null mice demonstrate impaired VEGF signaling and a marked reduction in arterial morphogenesis. Here we show that this occurs due to delayed trafficking of VEGFR2-containing endosomes that exposes internalized VEGFR2 to selective dephosphorylation by PTP1b on Y1175 site. Synectin involvement in VEGFR2 intracellular trafficking requires myosin-VI, and myosin-VI knockout in mice or knockdown in zebrafish phenocopy the synectin null phenotype. Silencing of PTP1b restores VEGFR2 activation and significantly recovers arterial morphogenesis in myosin-VI−/− knockdown zebrafish and synectin−/− mice. We conclude that activation of the VEGF-mediated arterial morphogenesis cascade requires phosphorylation of the VEGFR2 Y1175 site that is dependent on trafficking of internalized VEGFR2 away from the plasma membrane via a synectin-myosin-VI complex. This key event in VEGF signaling occurs at an intracellular site and is regulated by a novel endosomal trafficking-dependent process.

PHD2 serves as an oxygen sensor that rescues blood supply by regulating vessel formation and shape in case of oxygen shortage1–5. However, it is unknown whether PHD2 can influence arteriogenesis. Here we studied the role of PHD2 in collateral artery growth by using hindlimb ischaemia as a model, a process that compensates for the lack of blood flow in case of major arterial occlusion6–8. We show that Phd2 (also known as Egln1) haplodeficient (Phd2+/−) mice displayed preformed collateral arteries that preserved limb perfusion and prevented tissue necrosis in ischaemia. Improved arteriogenesis in Phd2+/− mice was due to an expansion of tissue-resident, M2-like macrophages9,10 and their increased release of arteriogenic factors, leading to enhanced smooth muscle cell (SMC) recruitment and growth. Both chronic and acute deletion of one Phd2 allele in macrophages was sufficient to skew their polarization towards a proarteriogenic phenotype. Mechanistically, collateral vessel preconditioning relied on the activation of canonical NF-κB pathway in Phd2+/− macrophages. These results unravel how PHD2 regulates arteriogenesis and artery homeostasis by controlling a specific differentiation state in macrophages and suggest new treatment options for ischaemic disorders.

SummaryNeuropilin 1 (NRP1) plays an important but ill-defined role in VEGF-A signaling and vascular morphogenesis. We show that mice with a knockin mutation that ablates the NRP1 cytoplasmic tail (Nrp1cyto) have normal angiogenesis but impaired developmental and adult arteriogenesis. The arteriogenic defect was traced to the absence of a PDZ-dependent interaction between NRP1 and VEGF receptor 2 (VEGFR2) complex and synectin, which delayed trafficking of endocytosed VEGFR2 from Rab5+ to EAA1+ endosomes. This led to increased PTPN1 (PTP1b)-mediated dephosphorylation of VEGFR2 at Y1175, the site involved in activating ERK signaling. The Nrp1cyto mutation also impaired endothelial tubulogenesis in vitro, which could be rescued by expressing full-length NRP1 or constitutively active ERK. These results demonstrate that the NRP1 cytoplasmic domain promotes VEGFR2 trafficking in a PDZ-dependent manner to regulate arteriogenic ERK signaling and establish a role for NRP1 in VEGF-A signaling during vascular morphogenesis.

Branching morphogenesis is a key process in the formation of vascular networks. To date, little is known regarding the molecular events regulating this process. We investigated the involvement of synectin in this process. In zebrafish embryos, synectin knockdown resulted in a hypoplastic dorsal aorta and hypobranched, stunted, and thin intersomitic vessels due to impaired migration and proliferation of angioblasts and arterial endothelial cells while not affecting venous development. Synectin(-/-) mice demonstrated decreased body and organ size, reduced numbers of arteries, and an altered pattern of arterial branching in multiple vascular beds while the venous system remained normal. Murine synectin(-/-) primary arterial, but not venous, endothelial cells showed decreased in vitro tube formation, migration, and proliferation and impaired polarization due to abnormal localization of activated Rac1. We conclude that synectin is involved in selective regulation of arterial, but not venous, growth and branching morphogenesis and that Rac1 plays an important role in this process.