Hydrogen Sulfide Attenuates Lymphedema Via the Induction of Lymphangiogenesis Through a PI3K/Akt-Dependent Mechanism.

Background Accumulating evidence suggests that hydrogen sulfide ( H2S ), 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 H2S 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 H2S 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 H2S 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, H2S donors could be used as therapeutics against refractory secondary lymphedema.

Adverse Effect of Circadian Rhythm Disorder on Reparative Angiogenesis in Hind Limb Ischemia.

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.

Important Role of Concomitant Lymphangiogenesis for Reparative Angiogenesis in Hindlimb Ischemia.

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No influence on tumor growth by intramuscular injection of adipose-derived regenerative cells: safety evaluation of therapeutic angiogenesis with cell therapy.

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 regarding 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 hindlimb ischemia (HLI) and simultaneous remote tumor implantation model. C57BL/6J background wild-type mice were injected with murine B16F10 melanoma cells on their back, 1 day before ischemic surgery. These mice were subjected to surgical unilateral hindlimb ischemia, followed by ADRC 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 with the PBS group. In addition, no distant metastases were detected in any of the mice regardless of the group. In conclusion, local implantation of ADRCs promotes angiogenesis in response to tissue ischemia in the hindlimb without promoting remote tumor growth and related angio/lymphangiogenesis. Therapeutic angiogenesis to the ischemic hindlimb using ADRCs seems to be safe regarding remote tumor growth.NEW & NOTEWORTHY In this study, we demonstrated that local injection of ADRCs can promote angiogenesis in response to tissue ischemia without promoting remote tumor growth in a mouse model. Our findings indicate that therapeutic angiogenesis to the ischemic hindlimb using ADRCs seems to be safe regarding remote tumor growth.