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

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.

Abstract 13133: Impact of Circadian Rhythm Disorders on Reparative Angiogenesis

Introduction: Circadian rhythm disorder seen in shift-worker or jet-lag is major social health concerns in advanced industrialized countries. The aim of this study was to examine if circadian rhythm disorders would influence on angiogenesis and blood perfusion recovery in a mouse model of hind limb ischemia (HLI). Methods and Results: First, we established a jet-lag model in C57BL/6J (wild type; WT) mice (8-10 weeks old, N=10 for each) using a light controlled isolation box. Mice were exposed to advanced 8-hr light phase once every 4 days in a jet-lag group as previously described. Conversely, control mice were kept a regular condition of LD 12:12 (12-hr light and 12-hr dark). Then, we surgically induced HLI in each group. The results showed that the condition jet-lag deteriorated capillary formation detected by CD31-immunohistochemistry at post-operative day (POD) 28 and tissue blood perfusion recovery demonstrated by laser Doppler perfusion imaging (LDPI) in HLI. The expression of clock genes (i.e. Clock, Bmal1, Per2, Cry1 and 2 ) in ischemic muscles were regulated by jet-lag condition at POD7.Next, we examined whether inhibition of clock gene had any effects on angiogenesis. For this study, we focused on Cryptochrome ( Cry ), which is well known as one of the core-loop forming clock genes producing circadian rhythm in mammals. Our loss-of-function study revealed that the abilities of proliferation, migration and tube formation were significantly inhibited by CRY1 and CRY2 double knockdown in HUVECs. Interestingly, although the knockdown of CRY1 and CRY2 changed the mRNA expression of PERIOD2 , it did not affect those of BMAL1 and CLOCK in HUVECs. Finally, we tested if Cry1 and Cry2 double knockout ( Cry1/2 -DKO) mice of HLI models displayed worse blood perfusion recoveries with deterioration of angiogenesis. Cry1/2 -DKO mice were reported to display circadian rhythm disorder in previous reports. As results, compared with control WT mice, Cry1/2 -DKO mice revealed suppressed capillary density and tissue blood perfusion recovery in HLI model. Conclusion: Our data suggest that a maintenance of circadian rhythm plays an important role in reparative angiogenesis of the tissue ischemia model.

Abstract TMP31: A Novel Shift of Notch Ligand, Dll4, Expression From Endothelial to Glial Cells in the Post-Stroke Brain May Contribute to Reparative Angiogenesis and Improved Functional Outcome

Delta-like 4 (Dll4) is a Notch receptor ligand classically thought to be expressed exclusively in endothelial cells. However, little is known about the cellular expression or functional role of Dll4 following ischemic stroke. Here, we present novel findings that Dll4 expression is shifted from endothelial cells to glial cells in the sub-acute stroke phase. Method: The permanent distal middle cerebral artery occlusion (dMCAO) model was performed to induce stroke. To investigate expression patterns of Dll4 protein and mRNA, immunostaining as well as single-cell RNA sequencing and single-cell fluorescent in situ hybridization were performed. To verify the role of endothelial Dll4 , tamoxifen-inducible, endothelial-specific Dll4 KO mice (Dll4iecKO) and Dll4 fl/fl (“floxed-only”) female mice (3 mos) were injected with tamoxifen (n=4) at 24 and 48 hrs post-stroke. To determine the effect of Notch inhibition at different time points, DAPT (100 mg/kg) or vehicle was administered at 24 or 72 hours post-stroke (n=4-5). Behavioral tests (foot fault) were conducted at Post Stroke Day (PSD) 1, 3, 7, and 14 and functional angiogenesis was assessed at PSD 14. Results: Elevated expression of Dll4 protein was observed in the microvasculature of the peri-infarct cortex (piCtx) as early as PSD 1. By PSD 3, however, we discovered Dll4 expression in microglia within the piCtx, with robust expression by PSD 14. Post-stroke deletion of endothelial Dll4 resulted in significant improvement in neurological function and increased capillary density in piCtx[l1] . DAPT given at 24 hours post-stroke improved outcome and capillary density; however, DAPT given at 72 hours (when microglia Dll4 expression is evident) appeared to worsen outcome. Conclusions: After stroke, expression of Dll4 shifts from exclusively endothelial (PSD 1) to mixed endothelial/microglial (PSD 3) to primarily microglial (PSD 14). Notch inhibition beginning at 24 hours post-stroke or endothelial-only deletion of Dll4 leads to increased angiogenesis and improved functional recovery. In contrast, Notch inhibition initiated at 72 hours post-stroke leads to worse outcome[l2] . These findings suggest a potential novel role of microglia in supporting reparative angiogenesis through Dll4/Notch signaling.

A Combinatorial Cell and Drug Delivery Strategy for Huntington’s Disease Using Pharmacologically Active Microcarriers and RNAi Neuronally-Committed Mesenchymal Stromal Cells

For Huntington’s disease (HD) cell-based therapy, the transplanted cells are required to be committed to a neuronal cell lineage, survive and maintain this phenotype to ensure their safe transplantation in the brain. We first investigated the role of RE-1 silencing transcription factor (REST) inhibition using siRNA in the GABAergic differentiation of marrow-isolated adult multilineage inducible (MIAMI) cells, a subpopulation of MSCs. We further combined these cells to laminin-coated poly(lactic-co-glycolic acid) PLGA pharmacologically active microcarriers (PAMs) delivering BDNF in a controlled fashion to stimulate the survival and maintain the differentiation of the cells. The PAMs/cells complexes were then transplanted in an ex vivo model of HD. Using Sonic Hedgehog (SHH) and siREST, we obtained GABAergic progenitors/neuronal-like cells, which were able to secrete HGF, SDF1 VEGFa and BDNF, of importance for HD. GABA-like progenitors adhered to PAMs increased their mRNA expression of NGF/VEGFa as well as their secretion of PIGF-1, which can enhance reparative angiogenesis. In our ex vivo model of HD, they were successfully transplanted while attached to PAMs and were able to survive and maintain this GABAergic neuronal phenotype. Together, our results may pave the way for future research that could improve the success of cell-based therapy for HDs.