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.

Eed/Sox2 regulatory loop controls ES cell self-renewal through histone methylation and acetylation.

Transcription factors and epigenetic modulators are involved in the maintenance of self-renewal in embryonic stem (ES) cells. Here, we demonstrate the existence of a regulatory loop in ES cells between Sox2, an indispensable transcription factor for self-renewal, and embryonic ectoderm development (Eed), an epigenetic modulator regulating histone methylation. We found that Sox2 and Eed positively regulate each other's expression. Interestingly, Sox2 overexpression suppressed the induction of differentiation-associated genes in Eed-deficient ES cells without restoring histone methylation. This Sox2-mediated suppression was prevented by knockdown of the histone acetyltransferase (HAT), Tip60 or Elp3, and Sox2 stimulated expression of these HATs. Furthermore, forced expression of either HAT resulted in repression of differentiation-associated genes in Eed-deficient cells. These results suggest that Sox2 overcame the phenotype of Eed-deficient ES cells by promoting histone acetylation. We also found that knockout of Eed and knockdown of these HATs synergistically enhanced the upregulation of differentiation-associated genes in ES cells. Taken together, our results suggest that the Eed/Sox2 regulatory loop contributes to the maintenance of self-renewal in ES cells by controlling histone methylation and acetylation.

Oral bioavailability of a noncoding RNA drug, TY1, that acts on macrophages.

All approved RNA therapeutics require parenteral delivery. Here we demonstrate an orally bioavailable formulation wherein synthetic noncoding (nc) RNA, packaged into lipid nanoparticles, is loaded into casein-chitosan (C2) micelles. We used the C2 formulation to deliver TY1, a 24-nucleotide synthetic ncRNA which targets the DNA damage response pathway in macrophages. C2-formulated TY1 (TY1C2) efficiently packages and protects TY1 against degradative enzymes. In healthy mice, oral TY1C2 was well-tolerated and nontoxic. Oral TY1C2 exhibited disease-modifying bioactivity in 2 models of tissue injury: 1) rat myocardial infarction, where a single oral dose of TY1C2 was cardioprotective, on par with intravenously-delivered TY1; and 2) mouse acute lung injury, where a single dose of TY1C2 attenuated pulmonary inflammation. Mechanistic dissection revealed that TY1C2 is not absorbed into the systemic circulation but is, instead, taken up by intestinal macrophages, namely those of the lamina propria and Peyer's patches. This route of absorption may rationalize why an antisense oligonucleotide against Factor VII, which acts on hepatocytes, is not effective when administered in the C2 formulation. Thus, some (but not all) ncRNA drugs are bioavailable when delivered by mouth. Oral RNA delivery and uptake, relying on uptake via the gastrointestinal immune system, has broad-ranging therapeutic implications.

A novel micropeptide, Slitharin, exerts cardioprotective effects in myocardial infarction.

PURPOSE: Micropeptides are an emerging class of proteins that play critical roles in cell signaling. Here, we describe the discovery of a novel micropeptide, dubbed slitharin (Slt), in conditioned media from Cardiosphere-derived cells (CDCs), a therapeutic cardiac stromal cell type. EXPERIMENTAL DESIGN: We performed mass spectrometry of peptide-enriched fractions from the conditioned media of CDCs and a therapeutically inert cell type (human dermal fibrobasts). We then evaluated the therapeutic capacity of the candidate peptide using an in vitro model of cardiomyocyte injury and a rat model of myocardial infarction. RESULTS: We identified a novel 24-amino acid micropeptide (dubbed Slitharin [Slt]) with a non-canonical leucine start codon, arising from long intergenic non-coding (LINC) RNA 2099. Neonatal rat ventricular myocytes (NRVMs) exposed to Slt were protected from hypoxic injury in vitro compared to a vehicle or scrambled control. Transcriptomic analysis of cardiomyocytes exposed to Slt reveals cytoprotective capacity, putatively through regulation of stress-induced MAPK-ERK. Slt also exerted cardioprotective effects in rats with myocardial infarction as shown by reduced infarct size 48 h post-injury. Conclusions and clinical relavance: Thus, Slt is a non-coding RNA-derived micropeptide, identified in the extracellular space, with a potential cardioprotective function.

Augmentation of DNA exonuclease TREX1 in macrophages as a therapy for cardiac ischemic injury.

Noncoding RNAs (ncRNAs) are increasingly recognized as bioactive. Here we report the development of TY1, a synthetic ncRNA bioinspired by a naturally-occurring human small Y RNA with immunomodulatory properties. TY1 upregulates TREX1, an exonuclease that rapidly degrades cytosolic DNA. In preclinical models of myocardial infarction (MI) induced by ischemia/reperfusion, TY1 reduced scar size. The cardioprotective effect of TY1 was abrogated by prior depletion of macrophages and mimicked by adoptive transfer of macrophages exposed either to TY1 or TREX1. Inhibition of TREX1 in macrophages blocked TY1 cardioprotection. Consistent with a central role for TREX1, TY1 attenuated DNA damage in the post-MI heart. This novel mechanism-pharmacologic upregulation of TREX1 in macrophages-establishes TY1 as the prototype for a new class of ncRNA drugs with disease-modifying bioactivity. One Sentence Summary: Upregulation of three prime exonuclease, TREX1, in macrophages enhances tissue repair post myocardial infarction.

Omentin Modulates Chronic Cardiac Remodeling After Myocardial Infarction.

Background: Omentin, a circulating adipokine, is downregulated in complications of obesity, including heart disease. Here, we investigated whether omentin modulates adverse cardiac remodeling in mice after myocardial infarction (MI). Methods and Results: Transgenic mice expressing the human omentin gene in fat tissue (OMT-Tg) and wild-type (WT) mice were subjected to permanent ligation of the left anterior descending coronary artery (LAD) to induce MI. OMT-Tg mice had a higher survival rate after permanent LAD ligation than WT mice. Moreover, OMT-Tg mice had lower heart weight/body weight (HW/BW) and lung weight/body weight (LW/BW) ratios at 4 weeks after coronary artery ligation compared with WT mice. OMT-Tg mice also showed decreased left ventricular diastolic diameter (LVDd) and increased fractional shortening (%FS) following MI. Moreover, an increase in capillary density in the infarct border zone and a decrease in myocardial apoptosis, myocyte hypertrophy, and interstitial fibrosis in the remote zone following MI, were more prevalent in OMT-Tg than WT mice. Finally, intravenous administration of adenoviral vectors expressing human omentin to WT mice after MI resulted in decreases in HW/BW, LW/BW, and LVDd, and an increase in %FS. Conclusions: Our findings document that human omentin prevents pathological cardiac remodeling after chronic ischemia, suggesting that omentin represents a potential therapeutic molecule for the treatment of ischemic heart disease.

TDO2-augmented fibroblasts secrete EVs enriched in immunomodulatory Y-derived small RNA.

Mounting evidence implicates extracellular vesicles (EVs) factors as mediators of cell therapy. Cardiosphere-derived cells are cardiac-derived cells with tissue reparative capacity. Activation of a downstream target of wnt/ß-catenin signalling, tryptophan 2,3 dioxygenase (TDO2) renders therapeutically inert skin fibroblasts cardioprotective. Here, we investigate the mechanism by which concentrated conditioned media from TDO2-augmented fibroblasts (TDO2-CCM) exert cardioprotective effects. TDO2-CCM is cardioprotective in a mouse model of MI compared to CCM from regular fibroblasts (HDF-CCM). Transcriptomic analysis of cardiac tissue at 24 h demonstrates broad suppression of inflammatory and cell stress markers in animals given TDO2-CCM compared to HDF-CCM or vehicle. Sequencing analysis of TDO2-EV RNA demonstrated abundance of a small Y-derived small RNA dubbed 'NT4'. Purification of TDO2-EVs by size-exclusion chromatography and RNAse protection assays demonstrated that NT4 is encapsulated inside EVs. Consistently with TDO2-CCM, macrophages exposed to NT4 showed suppression of the inflammatory and cell stress mediators, particularly p21/cdkn1a. NT4-depleted TDO2-CCM resulted in diminished immunomodulatory capacity. Finally, administration of NT4 alone was cardioprotective in an acute model of myocardial infarction. Taken together, these findings elucidate the mechanism by which TDO2 augmentation mediates potency in secreted EVs through enrichment of NT4 which suppresses upstream cell stress mediators including p21/cdkn1a.

Adipose-derived regenerative cells as a promising therapy for cardiovascular diseases: an overview.

The number of patients with ischemic cardiovascular diseases is significantly increasing as populations age. Therapeutic angiogenesis has been developed as a new treatment strategy for such patients. In recent years, the presence of mesenchymal stem cells in adipose tissues was reported, and regenerative medicine using these cells has attracted attention worldwide. In this review, we describe how the transplantation of adipose-derived regenerative cells enhances angiogenesis and tissue regeneration because of their multilineage potential and cytokine secretion. Then, the current status of therapeutic angiogenesis using adipose-derived regenerative cells in the field of cardiovascular medicine was also described. These cells present great advantages over bone marrow mononuclear cells, as these need easier, shorter, and less invasive preparations as well as less ethical concerns and immunological problems. The efficacy of adipose-derived regenerative cell transplantation in the treatment of various diseases was examined in several clinical trials with favorable results. Currently, a multicenter study of therapeutic angiogenesis using these cells is being conducted in patients with critical limb ischemia. In conclusion, we expect that this method will soon be established as a treatment for cardiovascular diseases that have been refractory to conventional treatments.

Zinc deficiency impairs ischemia-induced angiogenesis.

OBJECTIVE: Zinc is an important essential trace metal involved in many physiologic functions, and its deficiency can affect the development of multiple organs, including the vasculature. However, clarity is lacking regarding the effects of zinc deficiency in the regulation of angiogenesis. We investigated the effects of zinc deficiency on the revascularization process through animal experiments and examined the relationship between the circulating zinc levels and tissue blood perfusion in patients with chronic limb-threatening ischemia (CLTI). METHODS: Zinc-deficient mice and control wild-type mice had undergone surgery to create unilateral hindlimb ischemia. Next, we examined the relationship between the serum zinc levels and skin perfusion pressure (SPP) as an index of tissue blood perfusion in patients with CLTI. A total of 51 patients with CLTI who had been referred for de novo revascularization for CLTI due to arteriosclerosis obliterans at our hospital from May 2012 to March 2016 were enrolled. RESULTS: The zinc-deficient mice showed a significant reduction in blood flow recovery rates in the ischemic limb and capillary density in the ischemic adductor muscle fibers compared with the control wild-type mice. The zinc-deficient mice also showed increased reactive oxygen species production after hindlimb ischemia. Nicotinamide adenine dinucleotide phosphate oxidase inhibitors ameliorated the zinc deficient-induced impairment of revascularization. The serum zinc levels were positively associated with the SPP in the CLTI patients. Multivariate regression analysis also revealed that the serum zinc levels were significantly correlated with the SPP in patients with CLTI. CONCLUSIONS: Zinc deficiency impaired the rate of ischemia-induced revascularization through enhanced oxidative stress rates, suggesting that nutritional management for zinc sufficiency could be useful in CLTI prevention and treatment.

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.

Therapeutic angiogenesis using autologous adipose-derived regenerative cells in patients with critical limb ischaemia in Japan: a clinical pilot study.

Adipose-derived regenerative cell (ADRC) is a promising alternative source of autologous somatic stem cells for the repair of damaged tissue. This study aimed to assess the safety and feasibility of autologous ADRC implantation for therapeutic angiogenesis in patients with critical limb ischaemia (CLI). A clinical pilot study-Therapeutic Angiogenesis by Cell Transplantation using ADRCs (TACT-ADRC) study-was initiated in Japan. Adipose tissue was obtained by ordinary liposuction method. Isolated ADRCs were injected into the ischaemic limb. We performed TACT-ADRC procedure in five patients with CLI. At 6 months, no adverse events related to the TACT-ADRC were observed. No patients required major limb amputation, and ischaemic ulcers were partly or completely healed during the 6-month follow-up. In all cases, significant clinical improvements were seen in terms of rest pain and 6-min walking distance. Numbers of circulating CD34+ and CD133+ cells markers of progenitor cell persistently increased after ADRC implantation. The ratio of VEGF-A165b (an anti-angiogenic isoform of VEGF) to total VEGF-A in plasma significantly decreased after ADRC implantation. In vitro experiments, cultured with ADRC-conditioned media (CM) resulted in increased total VEGF-A and decreased VEGF-A165b in C2C12 cells, but not in macrophages. ADRC-CM also increased CD206+ cells expression and decreased TNF-α in macrophages. Autologous ADRC implantation was safe and effective in patients with CLI and could repair damaged tissue via its ability to promote angiogenesis and suppress tissue inflammation.

C1q/TNF-Related Protein 9 Promotes Revascularization in Response to Ischemia via an eNOS-Dependent Manner.

Strategies to promote revascularization are valuable for ischemic cardiovascular disease. Although C1q/TNF-related protein (CTRP) 9 is an adiponectin paralog with protective properties against cardiometabolic disorders, the role of endogenous CTRP9 in endothelial function is largely unknown. This study aimed to investigate the effects of CTRP9 on revascularization processes and dissected the potential mechanisms. CTRP9-knockout (KO) and wild-type (WT) mice were subjected to unilateral hindlimb ischemic surgery. CTRP9-KO mice exhibited impaired blood flow recovery and decreased capillary density in the ischemic limb compared with WT mice. In both CTRP9-KO and WT mice, systemic delivery of an adenoviral vector expressing CTRP9 (Ad-CTRP9) accelerated blood flow recovery. Treatment with recombinant CTRP9 protein increased network formation and migration of cultured human umbilical vein endothelial cells (HUVECs). CTRP9 promoted the phosphorylation of AMP-activated kinase (AMPK), Akt, and endothelial nitric oxide synthase (eNOS) in HUVECs. CTRP9-KO mice also showed reduced phosphorylation levels of AMPK, Akt, and eNOS in the ischemic limbs compared with WT mice. Furthermore, blockade of AMPK or Akt signaling pathway reversed the CTRP9-stimulated eNOS phosphorylation in HUVECs. Treatment with the NOS inhibitor significantly reduced CTRP9-stimulated network formation and migration of HUVECs. Of note, Ad-CTRP9 had no effects on blood flow of the ischemic limb in eNOS-KO mice. These results indicated that CTRP9 promotes endothelial cell function and ischemia-induced revascularization through the eNOS-dependent mechanism, suggesting that CTRP9 represents a target molecule for treatment of ischemic vascular diseases.

Adipolin/CTRP12 protects against pathological vascular remodelling through suppression of smooth muscle cell growth and macrophage inflammatory response.

AIMS: Secreted factors produced by adipose tissue are involved in the pathogenesis of cardiovascular disease. We previously identified adipolin, also known as C1q/TNF-related protein 12, as an insulin-sensitizing adipokine. However, the role of adipolin in vascular disease remains unknown. Here, we investigated whether adipolin modulates pathological vascular remodelling. METHODS AND RESULTS: Adipolin-knockout (APL-KO) and wild-type (WT) mice were subjected to wire-induced injury of the femoral artery. APL-KO mice showed increased neointimal thickening after vascular injury compared with WT mice, which was accompanied by an enhanced inflammatory response and vascular cell proliferation in injured arteries. Adipolin deficiency also led to a reduction in transforming growth factor-ß (TGF-ß) 1 protein levels in injured arteries. Treatment of cultured macrophages with adipolin protein led to a reduction in lipopolysaccharide-stimulated expression of inflammatory mediators, including tumour necrosis factor (TNF)-α, interleukin (IL) 6, and monocyte chemotactic protein (MCP)-1. These effects were reversed by inhibition of TGF-ß receptor II (TGF-ßRII)/Smad2 signalling. Adipolin also reduced platelet-derived growth factor (PDGF)-BB-stimulated proliferation of vascular smooth muscle cells (VSMCs) through a TGF-ßRII/Smad2-dependent pathway. Furthermore, adipolin treatment significantly increased TGF-ß1 concentration in media from cultured VSMCs and macrophages. CONCLUSION: These data indicate that adipolin protects against the development of pathological vascular remodelling by attenuating macrophage inflammatory responses and VSMC proliferation.