Arteriogenesis rather than unspecialized capillary expansion is critical for restoring effective circulation to compromised tissues in patients. Deciphering the origin and specification of arterial endothelial cells during embryonic development will shed light on the understanding of adult arteriogenesis. However, during early embryonic angiogenesis, the process of endothelial diversification and molecular events underlying arteriovenous fate settling remain largely unresolved in mammals. Here, we constructed the single-cell transcriptomic landscape of vascular endothelial cells (VECs) during the time window for the occurrence of key vasculogenic and angiogenic events in both mouse and human embryos. We uncovered two distinct arterial VEC types, the major artery VECs and arterial plexus VECs, and unexpectedly divergent arteriovenous characteristics among VECs that are located in morphologically undistinguishable vascular plexus intra-embryonically. Using computational prediction and further lineage tracing of venous-featured VECs with a newly developed Nr2f2CrexER mouse model and a dual recombinase-mediated intersectional genetic approach, we revealed early and widespread arterialization from the capillaries with considerable venous characteristics. Altogether, our findings provide unprecedented and comprehensive details of endothelial heterogeneity and lineage relationships at early angiogenesis stages, and establish a new model regarding the arteriogenesis behaviors of early intra-embryonic vasculatures.

Introduction: The beneficial effect of amarogentin in the management of osteoporosis was determined using in vivo and in vitro methods. Material and methods: Experimental osteoporosis was induced in rats via bilateral ovariectomy. Rats were then treated for 5 weeks with amarogentin (50 and 100 mg/kg, p.o.). The levels of several biochemical markers of bone resorption and formation as well as bone mineral density (BMD) were measured in the rat serum. Isolated rat bone tissues were analysed using western blot assays. In the in vitro study, MG63 human osteoblasts were treated with amarogentin (0-100 µg/ml), after which alkaline phosphatase activity and osteoblast proliferation were evaluated. Osteoblasts treated with amarogentin and inhibitors of extracellular signal-regulated kinase (ERK) were further examined via western blotting. Results: In the rat model of oestrogen-deficiency-induced osteoporosis, BMD was significantly enhanced (p < 0.01) and levels of inflammatory cytokines were reduced in amarogentin-treated animals vs. the controls. Amarogentin treatment also attenuated the altered levels of osteocalcin, C-telopeptide of type 1 collagen, procollagen type I N-terminal propeptide, and bone-specific alkaline phosphatase, and the altered expression of Akt, Nrf-2, ERK, and nuclear factor-κB p65 in the serum of rats with osteoporosis. In the in vitro study, amarogentin treatment enhanced alkaline phosphatase activity and osteoblast proliferation compared to the non-treated control. Amarogentin treatment alone enhanced the expression of pERK compared to treatment with an amarogentin + ERK inhibitor. Conclusions: Both the in vivo and the in vitro studies demonstrated the protective effect of amarogentin against oestrogen-deficiency-induced osteoporosis in rats. The mechanism seems to involve the amarogentin-mediated enhancement of osteoblast differentiation via the Nrf-2/MAPK/ERK signalling pathway.