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.

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.

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.

Comparison between unattended automated office blood pressure and conventional office blood pressure under the environment of health checkup among Japanese general population.

Unattended automated office blood pressure (AOBP) measurement has been endorsed as the preferred in-office measurement modality in recent Canadian and American clinical practice guidelines. However, the difference between AOBP and conventional office blood pressure (CBP) under the environment of a health checkup remains unclear. We aimed to identify the clinical significance of AOBP as compared to CBP under the environment of a health checkup. There were 491 participants (333 females, mean age of 62.5 years) who were at least 20 years old, including 179 participants who were previously diagnosed with hypertension. Mean AOBPs were 131.8 ± 20.9/76.6 ± 11.7 mm Hg, and CBPs were 135.6 ± 21.6/77.3 ± 11.5 mm Hg. There was a difference of 3.9 mm Hg in systolic blood pressure (SBP) and 0.8 mm Hg in diastolic BP between AOBP and CBP. In all participants, SBP and pulse pressure, as well as the white coat effect (WCE), increased with age. The cutoff value used was 140/90 mm Hg for CBP and 135/85 mm Hg for AOBP, and the prevalence of WCE and masked hypertension effect (MHE) was 12.4% and 14.1%, respectively. Even in a health checkup environment of the general population, there was a difference between the AOBP and CBP, and the WCE was observed more strongly in the elderly with a history of hypertension, suggesting that a combination of AOBP with CBP may be useful in detecting WCE and MHE in all clinical scenarios including health checkups, and help solve the "hypertension paradox" not only in Japan but in all over the world.

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.

Expression of granzyme B in human articular chondrocytes.

OBJECTIVE: To investigate the expression of granzyme B (GrB) in normal and rheumatoid arthritis (RA) articular cartilage, and to analyze the relationship between the expression of GrB and apoptotic chondrocytes in RA cartilage. METHODS: Normal cartilage samples were obtained from 9 resected joints and RA cartilage samples were obtained from 12 patients with RA during joint replacement surgery. Cartilage sections were analyzed by immunohistochemistry for the presence of GrB, and the mRNA expression of GrB in chondrocytes was analyzed by in situ hybridization and nonquantitative and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The expression of perforin (PFN) was also assessed. Apoptotic chondrocytes were detected using TUNEL staining and their morphology was examined using electron microscopy. RESULTS: The immunohistochemical analyses revealed GrB and PFN expression in normal chondrocytes and a larger number of GrB and PFN-positive chondrocytes in RA cartilage. In situ hybridization and RT-PCR confirmed the expression of GrB and PFN mRNA, and semiquantitative RT-PCR showed elevated concentrations of GrB and PFN expression in RA chondrocytes. The distribution of GrB and PFN-positive cells in the RA cartilage samples was similar to that of apoptotic cells. CONCLUSION: GrB and PFN expression is present in normal human articular chondrocytes and elevated in RA chondrocytes. The targets and precise functions of GrB expressed in chondrocytes remain to be determined, but GrB may be involved in the remodeling mechanism of matrix macromolecules and the endogenous degradation of RA cartilage.

Arthroscopic synovectomy of the elbow in rheumatoid arthritis.

BACKGROUND: The purpose of this study was to investigate the results of arthroscopic synovectomy for the treatment of elbows affected by rheumatoid arthritis. METHODS: Arthroscopic synovectomy was performed on twenty-nine elbows (twenty-seven patients) between 1984 and 1996. Twenty-one elbows (twenty patients), followed for a minimum of forty-two months, were evaluated clinically with use of the Mayo elbow performance score and radiographic findings. The mean duration of follow-up was ninety-seven months. With use of the system of Larsen et al., we classified all elbows into three groups--Grades 1 and 2, Grade 3, and Grade 4--according to the preoperative radiographic findings. These groups were then compared. RESULTS: The mean Mayo elbow performance score improved from 48.3 points preoperatively to 77.5 points (an excellent result in two elbows, a good result in thirteen, a fair result in six, and a poor result in none) at two years after the operation and 69.8 points (an excellent result in two elbows, a good result in seven, a fair result in seven, and a poor result in five) at the final follow-up evaluation. The mean score for pain improved from 9.3 points preoperatively to 31.4 points at two years after the operation and 27.9 points at the final follow-up evaluation. Clinically apparent synovitis recurred in five of the twenty-one elbows, and two of the five required total elbow arthroplasty. Among the three groups, only elbows with Larsen Grade-1 or 2 arthritis had a favorable long-term result with regard to total function. The postoperative results were unsatisfactory for Larsen Grade-4 elbows. CONCLUSIONS: Arthroscopic synovectomy in an elbow affected by rheumatoid arthritis is a reliable procedure that can alleviate pain. Our results suggest that one of the most favorable indications for arthroscopic synovectomy is a preoperative radiographic rating of Grade 1 or 2.