Background Circadian rhythm disorders, often seen in modern lifestyles, are a major social health concern. The aim of this study was to examine whether circadian rhythm disorders would influence angiogenesis and blood perfusion recovery in a mouse model of hind limb ischemia. Methods and Results A jet‐lag model was established in C57BL/6J mice using a light‐controlled isolation box. Control mice were kept at a light/dark 12:12 (12‐hour light and 12‐hour dark) condition. Concentrations of plasma vascular endothelial growth factor and circulating endothelial progenitor cells in control mice formed a circadian rhythm, which was diminished in the jet‐lag model ( P <0.05). The jet‐lag condition deteriorated tissue capillary formation ( P <0.001) and tissue blood perfusion recovery ( P <0.01) in hind limb ischemia, which was associated with downregulation of vascular endothelial growth factor expression in local ischemic tissue and in the plasma. Although the expression of clock genes (ie, Clock , Bmal1 , and Cry ) in local tissues was upregulated after ischemic injury, the expression levels of cryptochrome (Cry) 1 and Cry2 were inhibited by the jet‐lag condition. Next, Cry1 and Cry2 double‐knockout mice were examined for blood perfusion recoveries and a reparative angiogenesis. Cry1 and Cry2 double‐knockout mice revealed suppressed capillary density ( P <0.001) and suppressed tissue blood perfusion recovery ( P <0.05) in the hind limb ischemia model. Moreover, knockdown of CRY1/2 in human umbilical vein endothelial cells was accompanied by increased expression of WEE1 and decreased expression of HOXC5 . This was associated with decreased proliferative capacity, migration ability, and tube formation ability of human umbilical vein endothelial cells, respectively, leading to impairment of angiogenesis. Conclusions Our data suggest that circadian rhythm disorder deteriorates reparative ischemia‐induced angiogenesis and that maintenance of circadian rhythm plays an important role in angiogenesis.

Objective: Lymphatic vessels are distributed throughout the body and tightly collaborate with blood vessels to maintain tissue homeostasis. However, the functional roles of lymphangiogenesis in the process of reparative angiogenesis in ischemic tissues are largely unknown. Accordingly, we investigated potential roles of lymphangiogenesis using a mouse model of ischemia-induced angiogenesis. Approach and Results: Male C57BL/6J mice were subjected to unilateral hindlimb ischemia, in which not only angiogenesis but also lymphangiogenesis was induced. Next, the excessive and prolonged tissue edema model significantly deteriorated reparative angiogenesis and blood perfusion recovery in ischemic limbs. Finally, implantation of adipose-derived regenerative cells augmented ischemia-induced lymphangiogenesis, which was accompanied by reduced tissue edema and inflammation, resulting in improving reparative angiogenesis and blood perfusion recovery. In addition, inhibition of lymphangiogenesis by MAZ51, a specific VEGFR3 (vascular endothelial cell growth factor receptor 3) inhibitor, resulted in enhanced inflammatory cell infiltration, gene expression of TNF (tumor necrosis factor)-α, IL (interleukin)-1β, IL-6, TGF (transforming growth factor)-β, angiostatin, vasohibin, and endostatin, and tissue edema, resulting in reduced angiogenesis. Conclusions: The lymphatic system may have a clearance role of tissue edema and inflammation, which contribute to functional reparative angiogenesis in response to tissue ischemia. Modulation of lymphangiogenesis would become a novel therapeutic strategy for severe ischemic disease in addition to ordinary vascular intervention and therapeutic angiogenesis.

Objective Extracorporeal life support (ECLS) is effective for improving the survival rate of patients with refractory cardiac arrest (rCA). As little data are available regarding the impact of ECLS on a favorable neurological outcome, the predictors of a favorable neurological outcome were evaluated in this study. Methods Between January 2007 and August 2016, we retrospectively recruited patients with rCA caused by cardiac events treated with ECLS in our institute. A favorable neurological outcome was defined as a Glasgow-Pittsburgh cerebral performance category score 1 at discharge. The study endpoint was the clinical outcomes and predictors of favorable neurologic patients at discharge. Results During the study period, 67 patients with CA caused by cardiac events (acute coronary syndrome, 57 patients; idiopathic ventricular fibrillation, 10 patients) were included. Of these, 20 patients (29.9%) were classified into the favorable neurological group. No marked difference was observed in the patient characteristics between those with and without a favorable outcome except for in the time from CA to starting ECLS (ECLS initiation time). A short ECLS initiation time resulted in a favorable outcome (37.8±28.1 minutes vs. 53.6±30.7 minutes, p=0.05). The cut-off time of ECLS initiation was 46 minutes, which was prolonged by the temporary return of spontaneous circulation before ECLS [odds ratio (OR), 3.69; 95% confidence interval (CI), 1.34-10.19; p=0.01] and transfer to the angiographic room (OR, 4.07; 95% CI, 1.44-11.53, p=0.008). Conclusion The early initiation of ECLS (within 46 minutes) might be associated with a favorable neurological outcome for patients with rCA caused by cardiac events.

Therapeutic angiogenesis with autologous stem/progenitor cells is a promising novel strategy for treatment of severe ischemic diseases. Human clinical trials utilizing autologous adipose-derived regenerative cells (ADRCs) have not reported treatment-related critical adverse effects thus far. However, there is still a large knowledge gap whether treatment of ischemic diseases with angiogenic therapy using ADRCs would promote unfavorable angiogenesis associated with tumors in vivo. Herein, we addressed this clinical question using a mouse hind limb ischemia (HLI) and simultaneous remote tumor implantation model. C57BL/6J background wild-type mice were injected with murine B16F10 melanoma cells on their back, one day before ischemic surgery. These mice were subjected to surgical unilateral hindlimb ischemia, followed by ADRCs implantation or PBS injection into the hindlimb ischemic muscles on the next day. Intramuscular implantation of ADRCs enhanced tissue capillary density and blood flow examined by a laser Doppler blood perfusion analysis in hind limb. However, this therapeutic regimen for ischemic limb using ADRCs did not affect remote melanoma growth nor the density of its feeder artery, angiogenesis and lymphatic vessels compared to the PBS group. In addition, no distant metastases were detected in any of the mice regardless the group. In conclusion, local implantation of ADRCs promotes angiogenesis in response to tissue ischemia in the hind limb without promoting remote tumor growth and related angio/lymphangiogenesis. Therapeutic angiogenesis to the ischemic hind limb using ADRCs seems to be safe regarding remote tumor growth.

Background Accumulating evidence suggests that hydrogen sulfide ( H 2 S ), an endogenously produced gaseous molecule, plays a critical role in the regulation of cardiovascular homeostasis. However, little is known about its role in lymphangiogenesis. Thus, the current study aimed to investigate the involvement of H 2 S in lymphatic vessel growth and lymphedema resolution using a murine model and assess the underlying mechanisms. Methods and Results A murine model of tail lymphedema was created both in wild‐type mice and cystathionine γ‐lyase–knockout mice, to evaluate lymphedema up to 28 days after lymphatic ablation. Cystathionine γ‐lyase–knockout mice had greater tail diameters than wild‐type mice, and this phenomenon was associated with the inhibition of reparative lymphangiogenesis at the site of lymphatic ablation. In contrast, the administration of an H 2 S donor, diallyl trisulfide, ameliorated lymphedema by inducing the formation of a considerable number of lymphatic vessels at the injured sites in the tails. In vitro experiments using human lymphatic endothelial cells revealed that diallyl trisulfide promoted their proliferation and differentiation into tube‐like structures by enhancing Akt (protein kinase B) phosphorylation in a concentration‐dependent manner. The blockade of Akt activation negated the diallyl trisulfide–induced prolymphangiogenic responses in lymphatic endothelial cells. Furthermore, the effects of diallyl trisulfide treatment on lymphangiogenesis in the tail lymphedema model were also negated by the inhibition of phosphoinositide 3'‐kinase (P13K)/Akt signaling. Conclusions H 2 S promotes reparative lymphatic vessel growth and ameliorates secondary lymphedema, at least in part, through the activation of the Akt pathway in lymphatic endothelial cells. As such, H 2 S donors could be used as therapeutics against refractory secondary lymphedema.