Inducible deletion of microRNA activity in kidney mesenchymal cells exacerbates renal fibrosis.

MicroRNAs (miRNAs) are sequence-specific inhibitors of post-transcriptional gene expression. However, the physiological functions of these non-coding RNAs in renal interstitial mesenchymal cells remain unclear. To conclusively evaluate the role of miRNAs, we generated conditional knockout (cKO) mice with platelet-derived growth factor receptor-ß (PDGFR-ß)-specific inactivation of the key miRNA pathway gene Dicer. The cKO mice were subjected to unilateral ureteral ligation, and renal interstitial fibrosis was quantitatively evaluated using real-time polymerase chain reaction and immunofluorescence staining. Compared with control mice, cKO mice had exacerbated interstitial fibrosis exhibited by immunofluorescence staining and mRNA expression of PDGFR-ß. A microarray analysis showed decreased expressions of miR-9-5p, miR-344g-3p, and miR-7074-3p in cKO mice compared with those in control mice, suggesting an association with the increased expression of PDGFR-ß. An analysis of the signaling pathways showed that the major transcriptional changes in cKO mice were related to smooth muscle cell differentiation, regulation of DNA metabolic processes and the actin cytoskeleton, positive regulation of fibroblast proliferation and Ras protein signal transduction, and focal adhesion-PI3K/Akt/mTOR signaling pathways. Depletion of Dicer in mesenchymal cells may downregulate the signaling pathway related to miR-9-5p, miR-344g-3p, and miR-7074-3p, which can lead to the progression of chronic kidney disease. These findings highlight the possibility for future diagnostic or therapeutic developments for renal fibrosis using miR-9-5p, miR-344g-3p, and miR-7074-3p.

NG2-positive pericytes regulate homeostatic maintenance of slow-type skeletal muscle with rapid myonuclear turnover.

BACKGROUND: Skeletal muscle comprises almost 40% of the human body and is essential for movement, structural support and metabolic homeostasis. Size of multinuclear skeletal muscle is stably maintained under steady conditions with the sporadic fusion of newly produced myocytes to compensate for the muscular turnover caused by daily wear and tear. It is becoming clear that microvascular pericytes (PCs) exhibit myogenic activity. However, whether PCs act as myogenic stem cells for the homeostatic maintenance of skeletal muscles during adulthood remains uncertain. METHODS: We utilized PC-fused myofibers using PC-specific lineage tracing mouse (NG2-CreERT/Rosa-tdTomato) to observe whether muscle resident PCs have myogenic potential during daily life. Genetic PC deletion mouse model (NG2-CreERT/DTA) was used to test whether PC differentiates to myofibers for maintenance of muscle structure and function under homeostatic condition. RESULTS: Under steady breeding conditions, tdTomato-expressing PCs were infused into myofibers, and subsequently, PC-derived nuclei were incorporated into myofibers. Especially in type-I slow-type myofibers such as the soleus, tdTomato+ myofibers were already observed 3 days after PC labeling; their ratio reached a peak (approximately 80%) within 1 month and was maintained for more than 1 year. Consistently, the NG2+ PC-specific deletion induced muscular atrophy in a slow-type myofiber-specific manner under steady breeding conditions. The number of myonucleus per volume of each myofiber was constant during observation period. CONCLUSIONS: These findings demonstrate that the turnover of myonuclei in slow-type myofibers is relatively fast, with PCs acting as myogenic stem cells-the suppliers of new myonuclei under steady conditions-and play a vital role in the homeostatic maintenance of slow-type muscles.

Prostaglandin E2 mediates the late phase of ischemic preconditioning in the heart via its receptor subtype EP4.

Ischemic preconditioning (IPC) describes a phenomenon wherein brief ischemia of the heart induces a potent cardioprotective mechanism against succeeding ischemic insult. Cyclooxygenase-2 (COX-2), a rate-limiting enzyme in prostanoid biosynthesis, is upregulated in the ischemic heart and contributes to IPC. Prostaglandin E2 (PGE2) protects the heart from ischemia-reperfusion (I/R) injury via its receptor subtype EP4. We sought to clarify the role of the PGE2/EP4 system in the late phase of IPC. Mice were subjected to four IPC treatment cycles, consisting of 5 min of occlusion of the left anterior descending coronary artery (LAD). We found that COX-2 mRNA was significantly upregulated in wild-type hearts at 6 h after IPC treatment. Cardiac PGE2 levels at 24 h after IPC treatment were significantly increased in both wild-type mice and mice lacking EP4 (EP4-/-). At 24 h after IPC treatment, I/R injury was induced by 30 min of LAD occlusion followed by 2 h of reperfusion and the cardiac infarct size was determined. The infarct size was significantly reduced by IPC treatment in wild-type mice; a reduction was not observed in EP4-/- mice. AE1-329, an EP4 agonist, significantly reduced infarct size and significantly ameliorated deterioration of cardiac function in wild-type mice subjected to I/R without IPC treatment. Furthermore, AE1-329 significantly enhanced the I/R-induced activation of Akt, a pro-survival kinase. We demonstrated that the PGE2/EP4 system in the heart plays a critical role in the late phase of IPC, partly by augmenting Akt-mediated signaling. These findings clarify the mechanism of IPC and may contribute to the development of therapeutic strategies for ischemic heart disease.

Image-based crosstalk analysis of cell-cell interactions during sprouting angiogenesis using blood-vessel-on-a-chip.

BACKGROUND: Sprouting angiogenesis is an important mechanism for morphogenetic phenomena, including organ development, wound healing, and tissue regeneration. In regenerative medicine, therapeutic angiogenesis is a clinical solution for recovery from ischemic diseases. Mesenchymal stem cells (MSCs) have been clinically used given their pro-angiogenic effects. MSCs are reported to promote angiogenesis by differentiating into pericytes or other vascular cells or through cell-cell communication using multiple protein-protein interactions. However, how MSCs physically contact and move around ECs to keep the sprouting angiogenesis active remains unknown. METHODS: We proposed a novel framework of EC-MSC crosstalk analysis using human umbilical vein endothelial cells (HUVECs) and MSCs obtained from mice subcutaneous adipose tissue on a 3D in vitro model, microvessel-on-a-chip, which allows cell-to-tissue level study. The microvessels were fabricated and cultured for 10 days in a collagen matrix where MSCs were embedded. RESULTS: Immunofluorescence imaging using a confocal laser microscope showed that MSCs smoothed the surface of the microvessel and elongated the angiogenic sprouts by binding to the microvessel's specific microstructures. Additionally, three-dimensional modeling of HUVEC-MSC intersections revealed that MSCs were selectively located around protrusions or roots of angiogenic sprouts, whose surface curvature was excessively low or high, respectively. CONCLUSIONS: The combination of our microvessel-on-a-chip system for 3D co-culture and image-based crosstalk analysis demonstrated that MSCs are selectively localized to concave-convex surfaces on scaffold structures and that they are responsible for the activation and stabilization of capillary vessels.

Ninjurin1 Deletion in NG2-Positive Pericytes Prevents Microvessel Maturation and Delays Wound Healing.

The formation of mature vasculature through angiogenesis is essential for adequate wound healing, such that blood-borne cells, nutrients, and oxygen can be delivered to the remodeling skin area. Neovessel maturation is highly dependent on the coordinated functions of vascular endothelial cells and perivascular cells, namely pericytes (PCs). However, the underlying mechanism for vascular maturation has not been completely elucidated, and its role in wound healing remains unclear. In this study, we investigated the role of Ninjurin-1 (Ninj1), a new molecule mediating vascular maturation, in wound healing using an inducible PC-specific Ninj1 deletion mouse model. Ninj1 expression increased temporarily in NG2-positive PCs in response to skin injury. When tamoxifen treatment induced a decreased Ninj1 expression in PCs, the neovessels in the regenerating wound margins were structurally and functionally immature, but the total number of microvessels was unaltered. This phenotypic change is associated with a reduction in PC-associated microvessels. Wound healing was significantly delayed in the NG2-specific Ninj1 deletion mouse model. Finally, we showed that Ninj1 is a crucial molecule that mediates vascular maturation in injured skin tissue through the interaction of vascular endothelial cells and PCs, thereby inducing adequate and prompt wound healing.

Electrostatic interactions between single arginine and phospholipids modulate physiological properties of sarcoplasmic reticulum Ca2+-ATPase.

Arg324 of sarcoplasmic reticulum Ca2+-ATPase forms electrostatic interactions with the phosphate moiety of phospholipids in most reaction states, and a hydrogen bond with Tyr122 in other states. Using site-directed mutagenesis, we explored the functional roles of Arg324 interactions, especially those with lipids, which at first glance might seem too weak to modulate the function of such a large membrane protein. The hydrogen bond forms transiently and facilitates Ca2+ binding from the cytoplasmic side. The contributions of the electrostatic interactions to the reaction steps were quantified using a rate vs activity coefficient plot. We found that the interaction between Arg324 and lipids decreases the affinity for luminal Ca2+. The transformation rate of the phosphoenzyme intermediate is facilitated by the electrostatic interactions, and the function of these interactions depends not only on the type but also on the composition of the phospholipids. The properties observed in microsomes could not be reproduced with any single phospholipid, but with a mixture of phospholipids that mimics the native membrane. These results suggest the importance of swapping of the lipid partners of different headgroups in the reaction step. This study shows that Arg324 plays a role in the reaction cycle via complex intra-protein and protein-lipid interactions.

VEGF-Independent Angiogenic Factors: Beyond VEGF/VEGFR2 Signaling.

Tumors induce angiogenesis to acquire oxygen and nutrition from their adjacent microenvironment. Tumor angiogenesis has been believed to be induced primarily by the secretion of vascular endothelial growth factor-A (VEGF-A) from various tumors. VEGF-A binds to VEGF receptor 2 (VEGFR2), resulting in subsequent activation of cellular substances regulating cell proliferation, survival, and angiogenesis. Antiangiogenic therapies targeting the VEGF-A/VEGFR2 axis, including bevacizumab and ramucirumab, humanized monoclonal antibodies against VEGF-A and VEGFR2, respectively, have been proposed as a promising strategy aimed at preventing tumor growth, invasion, and metastasis. Phase III clinical trials using bevacizumab and ramucirumab have shown that not all tumor patients benefit from such antiangiogenic agents, and that some patients who initially benefit subsequently become less responsive to these antibodies, suggesting the possible existence of VEGF-independent angiogenic factors. In this review, we focus on VEGF-independent and VEGFR2-dependent tumor angiogenesis, as well as VEGFR2-independent tumor angiogenesis. Additionally, we discuss VEGF-independent angiogenic factors which have been reported in previous studies. Various molecular targeting drugs are currently being evaluated as potential antitumor therapies. We expect that precision medicine will permit the development of innovative antiangiogenic therapies targeting individual angiogenic factors selected on the basis of the genetic screening of tumors.

Sarcopenia-derived exosomal micro-RNA 16-5p disturbs cardio-repair via a pro-apoptotic mechanism in myocardial infarction in mice.

Sarcopenia is a pathophysiological malfunction induced by skeletal muscle atrophy. Several studies reported an association between sarcopenia-induced cardiac cachexia and poor prognosis in heart disease. However, due to lack of an established animal models, the underlying mechanism of disturbed cardiac repair accompanied with sarcopenia remains poorly understood. Here, we developed a novel sarcopenia-induced cardiac repair disturbance mouse model induced by tail suspension (TS) after cardiac ischemia and reperfusion (I/R). Importantly, we identified a specific exosomal-microRNA marker, miR-16-5p, in the circulating exosomes of I/R-TS mice. Of note, sarcopenia after I/R disturbed cardiac repair and raised the level of circulating-exosomal-miR-16-5p secreting from both the atrophic limbs and heart of TS mice. Likewise, miR-16-5p mimic plasmid disturbed cardiac repair in I/R mice directly. Additionally, in neonatal rat ventricular myocytes (NRVMs) cultured in vitro under hypoxic conditions in the presence of a miR-16-5p mimic, we observed increased apoptosis through p53 and Caspase3 upregulation, and also clarified that autophagosomes were decreased in NRVMs via SESN1 transcript interference-mediated mTOR activation. In conclusion, we show the pro-apoptotic effect of sarcopenia-derived miR-16-5p, which may be behind the exacerbation of myocardial infarction. Therefore, miR-16-5p can be a novel therapeutic target in the context of cardiac repair disturbances in sarcopenia-cachexia.

Pericyte-specific deletion of ninjurin-1 induces fragile vasa vasorum formation and enhances intimal hyperplasia of injured vasculature.

Adventitial abnormalities including enhanced vasa vasorum malformation are associated with development and vulnerability of atherosclerotic plaque. However, the mechanisms of vasa vasorum malformation and its role in vascular remodeling have not been fully clarified. We recently reported that ninjurin-1 (Ninj1) is a crucial adhesion molecule for pericytes to form matured neovessels. The purpose is to examine if Ninj1 regulates adventitial angiogenesis and affects the vascular remodeling of injured vessels using pericyte-specific Ninj1 deletion mouse model. Mouse femoral arteries were injured by insertion of coiled wire. Four weeks after vascular injury, fixed arteries were decolorized. Vascular remodeling, including intimal hyperplasia and adventitial microvessel formation were estimated in a three-dimensional view. Vascular fragility, including blood leakiness was estimated by extravasation of fluorescein isothiocyanate (FITC)-lectin or FITC-dextran from microvessels. Ninj1 expression was increased in pericytes in response to vascular injury. NG2-CreER/Ninj1loxp mice were treated with tamoxifen (Tam) to induce deletion of Ninj1 in pericyte (Ninj1 KO). Tam-treated NG2-CreER or Tam-nontreated NG2-CreER/Ninj1loxp mice were used as controls. Intimal hyperplasia was significantly enhanced in Ninj1 KO compared with controls. Vascular leakiness was significantly enhanced in Ninj1 KO. In Ninj1 KO, the number of infiltrated macrophages in adventitia was increased, along with the expression of inflammatory cytokines. In conclusion, deletion of Ninj1 in pericytes induces the immature vasa vasorum formation of injured vasculature and exacerbates adventitial inflammation and intimal hyperplasia. Thus, Ninj1 contributes to the vasa vasorum maturation in response to vascular injury and to reduction of vascular remodeling.NEW & NOTEWORTHY Although abnormalities of adventitial vasa vasorum are associated with vascular remodeling such as atherosclerosis, the mechanisms of vasa vasorum malformation and its role in vascular remodeling have not been fully clarified. The present study provides a line of novel evidence that ninjurin-1 contributes to adventitial microvascular maturation during vascular injury and regulates vascular remodeling.

EphA7+ perivascular cells as myogenic and angiogenic precursors improving skeletal muscle regeneration in a muscular dystrophic mouse model.

Skeletal muscle has a capacity for muscular regeneration mediated by satellite cells (SCs) and non-SCs. Although it is proposed that non-SCs are attractive therapeutic targets for dystrophies, the biological properties of these cells remain unclear. We have recently identified novel multipotent pericytes (PCs), capillary stem cells (CapSCs) derived from the microvasculature. In the present study, we determined if CapSCs contributed to myogenic regeneration using muscular dystrophy mouse model. CapSCs were isolated as EphA7+NG2+PCs from the subcutaneous adipose tissues of GFP-transgenic mice. Co-culture with C2C12 myoblast cells showed that CapSCs effectively enhanced myogenesis as compared to controls including EphA7- PCs and adipose stromal cells (ASCs). CapSCs transplanted in cardiotoxin-injured gastrocnemius muscles were well differentiated into both muscle fibers and microvessels, as compared to controls. At three weeks after cell-transplantation into the limbs of the mdx/utrn-/-mouse, CapSCs increased the number of GFP+myofibers along with dystrophin expression and the area size of myofibers, and also enhanced the muscular mass and its performance, assessed by treadmill test as compared to controls. In conclusion, CapSCs have potent myogenic regeneration capacity and improved the pathological condition in a muscular dystrophy mouse. Thus, CapSCs are an attractive cellular source in regenerative therapy for muscular dystrophy.

Capillary-resident EphA7+ pericytes are multipotent cells with anti-ischemic effects through capillary formation.

The presence of pericytes (PCs) with multipotency and broad distribution along capillary suggests that microvasculature plays a role not only as a duct for blood fluid transport but also as a stem cell niche that contributes to tissue maintenance and regeneration. The lack of an appropriate marker for multipotent PCs still limits our understanding of their pathophysiological roles. We identified the novel marker EphA7 to detect multipotent PCs using microarray analysis of an immortalized PC library. PCs were isolated from microvessels of mouse subcutaneous adipose tissues, then EphA7+ PCs called capillary stem cells (CapSCs) were separated from EphA7- control PCs (ctPCs) using fluorescence-activated cell sorting system. CapSCs had highly multipotency that enabled them to differentiate into mesenchymal and neuronal lineages compared with ctPCs. CapSCs also differentiated into endothelial cells and PCs to form capillary-like structures by themselves. Transplantation of CapSCs into ischemic tissues significantly improved blood flow recovery in hind limb ischemia mouse model due to vascular formation compared with that of ctPCs and adipose stromal cells. These data demonstrate that EphA7 identifies a subpopulation of multipotent PCs that have high angiogenesis and regenerative potency and are an attractive target for regenerative therapies.

Ninjurin 1 mediates peripheral nerve regeneration through Schwann cell maturation of NG2-positive cells.

Ninjurin 1 (Ninj1) is identified as a peripheral nerve injury-induced protein. However, the role of Ninj1 in nerve regeneration is unclear. Schwann cells (SCs) and microvasculature are critical for peripheral nerve regeneration. SCs precursors and microvascular pericytes (PCs), which are nerve/glial antigen 2 (NG2)-positive cells are observed in peripheral nervous system. In this study, we investigated the role of Ninj1 in peripheral nerve regeneration using NG2+cell-specific inducible deletion of Ninj1 mouse model. The number of NG2+cells, which were associated with and without microvessels was increased after sciatic nerve crush injury. There was a significant increase in the expression of Ninj1 and EphA7 in the injured nerve tissue. This increase was mostly observed in NG2+cells. Genetic tracing of NG2+cells was performed using tamoxifen (Tam) treatment on NG2CreERT:R26R-tdTomato mice. The sciatic nerve was injured following the Tam-treatment, then tdTomato-expressing SCs were mostly observed in regenerated SCs at 21 days after nerve injury. Ninj1 gene knockout (Ninj1 KO) in NG2+cells was induced using NG2CreERT:Ninj1loxp mice. Tam-treated-NG2CreERT or Tam-nontreated NG2CreERT:Ninj1loxp mice were used as controls. Following Tam-treatment, the sciatic nerve in each group was injured. Ninj1KO significantly attenuated the expression of the myelin binding protein (MBP) as well as the number of myelinated axons. The expression of MBP in cultured SCs was significantly reduced by SiRNA-mediated Ninj1 knockdown (KD). Ninj1KD also attenuated the differentiation of SCs by isolated EphA7+multipotent PCs. The current data indicate that Ninj1 plays a vital role in peripheral nerve regeneration. This is observed particularly in the myelination process of NG2+cells including SCs precursors and multipotent PCs.

The antioxidant and DNA-repair enzyme apurinic/apyrimidinic endonuclease 1 limits the development of tubulointerstitial fibrosis partly by modulating the immune system.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein that controls the cellular response to oxidative stress and possesses DNA-repair functions. It has important roles in the progression and outcomes of various diseases; however, its function and therapeutic prospects with respect to kidney injury are unknown. To study this, we activated APE1 during kidney injury by constructing an expression vector (pCAG-APE1), using an EGFP expression plasmid (pCAG-EGFP) as a control. We performed unilateral ureteral obstruction (UUO) as a model of tubulointerstitial fibrosis on ICR mice before each vector was administrated via retrograde renal vein injection. In this model, pCAG-APE1 injection did not produce any adverse effects and significantly reduced histological end points including fibrosis, inflammation, tubular injury, and oxidative stress, as compared to those parameters after pCAG-EGFP injection. qPCR analysis showed significantly lower expression of Casp3 and inflammation-related genes in pCAG-APE1-injected animals compared to those in pCAG-EGFP-injected UUO kidneys. RNA-Seq analyses showed that the major transcriptional changes in pCAG-APE1-injected UUO kidneys were related to immune system processes, metabolic processes, catalytic activity, and apoptosis, leading to normal kidney repair. Therefore, APE1 suppressed renal fibrosis, not only via antioxidant and DNA-repair functions, but also partly by modulating the immune system through multiple pathways including Il6, Tnf, and chemokine families. Thus, therapeutic APE1 modulation might be beneficial for the treatment of renal diseases.

Pericyte-Specific Ninjurin1 Deletion Attenuates Vessel Maturation and Blood Flow Recovery in Hind Limb Ischemia.

Objective- Angiogenesis, entire step from endothelial cells (ECs) sprouts to vascular maturation, is a critical response to ischemia. To form functional mature vessels, interactions between ECs and pericytes are essential. Ninj1 (ninjurin1) is an adhesion molecule that contributes to the pathogenesis of neuroinflammation. We recently demonstrated that Ninj1 is expressed in pericytes during angiogenesis. However, the role of Ninj1 in angiogenesis under pathophysiological ischemic conditions has not yet been elucidated. Approach and Results- Ninj1 was detected in microvessels, and its expression was enhanced in ischemic tissues after mouse hindlimb ischemia. Knockdown of Ninj1 was performed by injection of biodegradable microspheres releasing Ninj1-small interfering RNA into muscle tissues. Alternatively, pericyte-specific Ninj1 knockout was induced by tamoxifen treatment of NG2-CreERT/Ninj1-flox mice. Ninj1 knockdown/knockout reduced the formation of blood-circulating functional vessels among total CD31+ microvessels within ischemic tissues and subsequently attenuated color Doppler-assessed blood flow recovery. Ninj1 overexpression enhanced expression of Anpt (angiopoietin) 1, whereas Ninj1 knockdown enhanced the endogenous Anpt1 antagonist, Anpt2 expression in pericytes and inhibited the association of pericytes with ECs and subsequent formation of capillary-like structure, that is, EC tube surrounded with pericytes in 3-dimensional gel culture. Conclusions- Our data demonstrate that Ninj1 is involved in the formation of functional matured vessels through the association between pericytes and ECs, resulting in blood flow recovery from ischemia. These findings further the current our understanding of vascular maturation and may support the development of therapeutics for ischemic diseases.

A 3D in vitro pericyte-supported microvessel model: visualisation and quantitative characterisation of multistep angiogenesis.

Angiogenesis, which refers to the formation of new blood vessels from already existing vessels, is a promising therapeutic target and a complex multistep process involving many different factors. Pericytes (PCs) are attracting attention as they are considered to make significant contributions to the maturation and stabilisation of newly formed vessels, although not much is known about the precise mechanisms involved. Since there is no single specific marker for pericytes, in vivo models may complicate PC identification and the study of PCs in angiogenesis would benefit from in vitro models recapitulating the interactions between PCs and endothelial cells (ECs) in a three-dimensional (3D) configuration. In this study, a 3D in vitro co-culture microvessel model incorporating ECs and PCs was constructed by bottom-up tissue engineering. Angiogenesis was induced in the manner of sprout formation by the addition of a vascular endothelial cell growth factor. It was found that the incorporation of PCs prevented expansion of the parent vessel diameter and enhanced sprout formation and elongation. Physical interactions between ECs and PCs were visualised by immunostaining and it disclosed that PCs covered the EC monolayer from its basal side in the parent vessel as well as angiogenic sprouts. Furthermore, the microvessels were visualized in 3D by using a non-invasive optical coherence tomography (OCT) imaging system and sprout features were quantitatively assessed. It revealed that the sprouts in EC-PC co-culture vessels were longer and tighter than those in EC mono-culture vessels. The combination of the microvessel model and the OCT system analysis can be useful for the visualisation and demonstration of the multistep process of angiogenesis, which incorporates PCs.

Apoptosis-Resistant Cardiac Progenitor Cells Modified With Apurinic/Apyrimidinic Endonuclease/Redox Factor 1 Gene Overexpression Regulate Cardiac Repair After Myocardial Infarction.

UNLABELLED: : Overcoming the insufficient survival of cell grafts is an essential objective in cell-based therapy. Apurinic/apyrimidinic endonuclease/redox factor 1 (APE1) promotes cell survival and may enhance the therapeutic effect of engrafted cells. The aim of this study is to determine whether APE1 overexpression in cardiac progenitor cells (CPCs) could ameliorate the efficiency of cell-based therapy. CPCs isolated from 8- to 10-week-old C57BL/6 mouse hearts were infected with retrovirus harboring APE1-DsRed (APE1-CPC) or a DsRed control (control-CPC). Oxidative stress-induced apoptosis was then assessed in APE1-CPCs, control-CPCs, and neonatal rat ventricular myocytes (NRVMs) cocultured with these CPCs. This analysis revealed that APE1 overexpression inhibited CPC apoptosis with activation of transforming growth factor ß-activated kinase 1 (TAK1) and nuclear factor (NF)-κB. In the coculture model, NRVM apoptosis was inhibited to a greater extent in the presence of APE1-CPCs compared with control-CPCs. Moreover, the number of surviving DsRed-positive CPC grafts was significantly higher 7 days after the transplant of APE1-CPCs into a mouse myocardial infarction model, and the left ventricular ejection fraction showed greater improvement with attenuation of fibrosis 28 days after the transplant of APE1-CPCs compared with control-CPCs. Additionally, fewer inflammatory macrophages and a higher percentage of cardiac α-sarcomeric actinin-positive CPC-grafts were observed in mice injected with APE1-CPCs compared with control-CPCs after 7 days. In conclusion, antiapoptotic APE1-CPC graft, which increased TAK1-NF-κB pathway activation, survived effectively in the ischemic heart, restored cardiac function, and reduced cardiac inflammation and fibrosis. APE1 overexpression in CPCs may serve as a novel strategy to improve cardiac cell therapy. SIGNIFICANCE: Improving the survival of cell grafts is essential to maximize the efficacy of cell therapy. The authors investigated the role of APE1 in CPCs under ischemic conditions and evaluated the therapeutic efficacy of transplanted APE1-overexpressing CPCs in a mouse model of myocardial infarction. APE1 hindered apoptosis in CPC grafts subjected to oxidative stress caused in part by increased TAK1-NF-κB pathway activation. Furthermore, APE1-CPC grafts that effectively survived in the ischemic heart restored cardiac function and attenuated fibrosis through pleiotropic mechanisms that remain to be characterized. These findings suggest that APE1 overexpression in CPCs may be a novel strategy to reinforce cardiac cell therapy.

Association between microalbuminuria predicting in-stent restenosis after myocardial infarction and cellular senescence of endothelial progenitor cells.

OBJECTIVE: Relationship between microalbuminuria and worse outcome of coronary artery disease patients is discussed, but its underlying pathophysiological mechanism remains unclear. We investigated the role of microalbuminuria to the function of endothelial progenitor cells (EPCs), that might affect to outcome of acute myocardial infarction (AMI) patients. METHODS: Forty-five AMI patients were divided into two groups according to their urinary albumin excretion: normal (n = 24) and microalbuminuria (>30 mg/day, n = 21). At day-2 and day-7 after AMI onset, circulating-EPCs (CD34+ Flk1+) were quantified by flow cytometry. The number of lectin-acLDL-positive cultured-EPCs immobilized on fibronectin was determined. To assess the cellular senescence of cultured-EPCs, the expression level of sirtuin-1 mRNA and the number of SA-ß-gal positive cell were evaluated. Angiographic late in-stent loss after percutaneous coronary intervention (PCI) was evaluated at a six-month follow-up. RESULTS: No significant differences in coronary risk and the extent of myocardial damage were observed between the two groups. Late in-stent loss at the six-month follow-up was significantly higher in the microalbuminuria group (normal:microalbuminuria = 0.76±0.34:1.18±0.57 mm, p=0.021). The number of circulating-EPCs was significantly increased in microalbuminuria group at day-7, however, improved adhesion of EPCs was observed in normal group but not in microalbuminuria group from baseline to day-7 (+3.1±8.3:-1.3±4.4%: p<0.05). On the other hand, in microalbuminuria group at day-7, the level of sirtuin-1 mRNA expression of cultured-EPCs was significantly decreased (7.1±8.9:2.5±3.7 fold, p<0.05), which was based on the negative correlation between the level of sirtuin-1 mRNA expression and the extent of microalbuminuria. The ratio of SA-ß-gal-positive cells in microalbuminuria group was increased compared to that of normal group. CONCLUSIONS: Microalbuminuria in AMI patients is closely associated with functional disorder of EPCs via cellular senescence, that predicts the aggravation of coronary remodeling after PCI.

Ninjurin1 is a novel factor to regulate angiogenesis through the function of pericytes.

BACKGROUND: Capillary pericytes (cPCs), the mural cells of microvessels, play an important role in the formation and maintenance of microvessels; however, little is known about the mechanisms of how cPCs regulate angiogenesis. To identify factors that modulate cPC function, genes whose levels were altered in cPCs during neovessel formation were identified through a microarray screen. METHODS AND RESULTS: Ninjurin1 (nerve injury-induced protein, Ninj1) was selected as a candidate factor for angiogenesis regulation. Ninj1 was expressed in capillary cells including endothelial cells (cECs) and was expressed at a higher level in cPCs. Hypoxia induced the gene expression of Ninj1 in addition of vascular endothelial growth factor (VEGF) in cPCs. When cPCs were co-incubated with a thoracic aorta in a three-dimensional Matrigel system, the length of the EC-tubes sprouting from the aorta was increased. Small interfering RNA-mediated downregulation of Ninj1 in cPCs enhanced these cPCs-mediated angiogenic effects, whereas overexpression of Ninj1 attenuated their effects. The production of angiogenic growth factors, such as VEGF and angiopoietin 1, by cPCs was enhanced by the downregulation of Ninj1, and reduced by the overexpression of Ninj1. CONCLUSIONS: Ninj1 is a novel regulator for the angiogenic effect of PCs. Specifically, Ninj1 negatively regulates the formation of neovessels, that is, the EC-tube, by reducing the trophic effects of cPCs.

Prostaglandin I2 analog suppresses lung metastasis by recruiting pericytes in tumor angiogenesis.

Prostaglandin I2 (PGI2) agonist has been reported to reduce tumor metastasis by modifying tumor angiogenesis; however, the mechanisms of how PGI2 affects the endothelial cells or pericytes in tumor vessel maturation are still unclear. The purpose of this study was to clarify the effects of PGI2 on tumor metastasis in a mouse lung metastasis model using Lewis lung carcinoma (LLC) cells. The mice were treated continuously with beraprost sodium (BPS), a PGI2 analog, for 3 weeks and then examined for lung metastases. The number and size of lung metastases were decreased significantly by BPS treatment. In addition, scanning electron microscopy and immunohistochemistry revealed that BPS increased the number of tumor­associated pericytes and improved intratumor hypoxia. Collectively, this study suggests that BPS attenuated vascular functional maturation in metastatic tumors.

Immortalized multipotent pericytes derived from the vasa vasorum in the injured vasculature. A cellular tool for studies of vascular remodeling and regeneration.

Adventitial microvessels, vasa vasorum in the vessel walls, have an active role in the vascular remodeling, although its mechanisms are still unclear. It has been reported that microvascular pericytes (PCs) possess mesenchymal plasticity. Therefore, microvessels would serve as a systemic reservoir of stem cells and contribute to the tissues remodeling. However, most aspects of the biology of multipotent PCs (mPCs), in particular of pathological microvessels are still obscure because of the lack of appropriate methods to detect and isolate these cells. In order to examine the characteristics of mPCs, we established immortalized cells residing in adventitial capillary growing at the injured vascular walls. We recently developed in vivo angiogenesis to observe adventitial microvessels using collagen-coated tube (CCT), which also can be used as an adventitial microvessel-rich tissue. By using the CCT, CD146- or NG2-positive cells were isolated from the adventitial microvessels in the injured arteries of mice harboring a temperature-sensitive SV40 T-antigen gene. Several capillary-derived endothelial cells (cECs) and PCs (cPCs) cell lines were established. cECs and cPCs maintain a number of key endothelial and PC features. Co-incubation of cPCs with cECs formed capillary-like structure in Matrigel. Three out of six cPC lines, termed capillary mPCs demonstrated both mesenchymal stem cell- and neuronal stem cell-like phenotypes, differentiating effectively into adipocytes, osteoblasts, as well as schwann cells. mPCs differentiated to ECs and PCs, and formed capillary-like structure on their own. Transplanted DsRed-expressing mPCs were resident in the capillary and muscle fibers and promoted angiogenesis and myogenesis in damaged skeletal muscle. Adventitial mPCs possess transdifferentiation potential with unique phenotypes, including the reconstitution of capillary-like structures. Their phenotype would contribute to the pathological angiogenesis associated with vascular remodeling. These cell lines also provide a reproducible cellular tool for high-throughput studies on angiogenesis, vascular remodeling, and regeneration as well.