Maki Kabara

The intrinsic prostaglandin E2–EP4 system of the renal tubular epithelium limits the development of tubulointerstitial fibrosis in mice

Inflammatory responses in the kidney lead to tubulointerstitial fibrosis, a common feature of chronic kidney diseases. Here we examined the role of prostaglandin E(2) (PGE(2)) in the development of tubulointerstitial fibrosis. In the kidneys of wild-type mice, unilateral ureteral obstruction leads to progressive tubulointerstitial fibrosis with macrophage infiltration and myofibroblast proliferation. This was accompanied by an upregulation of COX-2 and PGE(2) receptor subtype EP(4) mRNAs. In the kidneys of EP(4) gene knockout mice, however, obstruction-induced histological alterations were significantly augmented. In contrast, an EP(4)-specific agonist significantly attenuated these alterations in the kidneys of wild-type mice. The mRNAs for macrophage chemokines and profibrotic growth factors were upregulated in the kidneys of wild-type mice after ureteral obstruction. This was significantly augmented in the kidneys of EP(4)-knockout mice and suppressed by the EP(4) agonist but only in the kidneys of wild-type mice. Notably, COX-2 and MCP-1 proteins, as well as EP(4) mRNA, were localized in renal tubular epithelial cells after ureteral obstruction. In cultured renal fibroblasts, another EP(4)-specific agonist significantly inhibited PDGF-induced proliferation and profibrotic connective tissue growth factor production. Hence, an endogenous PGE(2)-EP(4) system in the tubular epithelium limits the development of tubulointerstitial fibrosis by suppressing inflammatory responses.

Nerve growth factor stimulates regeneration of perivascular nerve, and induces the maturation of microvessels around the injured artery.

An immature vasa vasorum in the adventitia of arteries has been implicated in induction of the formation of unstable atherosclerotic plaques. Normalization/maturation of the vasa vasorum may be an attractive therapeutic approach for arteriosclerotic diseases. Nerve growth factor (NGF) is a pleotropic molecule with angiogenic activity in addition to neural growth effects. However, whether NGF affects the formation of microvessels in addition to innervation during pathological angiogenesis is unclear. In the present study, we show a new role for NGF in neovessels around injured arterial walls using a novel in vivo angiogenesis assay. The vasa vasorum around arterial walls was induced to grow using wire-mediated mouse femoral arterial injury. When collagen-coated tube (CCT) was placed beside the injured artery for 7-14 days, microvessels grew two-dimensionally in a thin layer on the CCT (CCT-membrane) in accordance with the development of the vasa vasorum. The perivascular nerve was found at not only arterioles but also capillaries in the CCT-membrane. Biodegradable hydrogels containing VEGF and NGF were applied around the injured artery/CCT. VEGF significantly increased the total length and instability of microvessels within the CCT-membrane. In contrast, NGF induced regeneration of the peripheral nerve around the microvessels and induced the maturation and stabilization of microvessels. In an ex vivo nerve-free angiogenesis assay, although NGF potentially stimulated vascular sprouting from aorta tissues, no effects of NGF on vascular maturation were observed. These data demonstrated that NGF had potent angiogenic effects on the microvessels around the injured artery, and especially induced the maturation/stabilization of microvessels in accordance with the regeneration of perivascular nerves.

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.

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.

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

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 β‐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.

Minimal Change Nephrotic Syndrome Associated with Gefitinib and a Successful Switch to Erlotinib

Minimal change nephrotic syndrome (MCNS) is a common form of nephrotic syndrome (NS). We herein present the case of a 57-year-old woman with advanced lung adenocarcinoma treated with the tyrosine kinase inhibitor (TKI) gefitinib who developed NS. A renal biopsy revealed minor glomerular abnormalities, and the patients symptoms improved exclusively with the discontinuation of gefitinib. Therefore, we diagnosed her with MCNS associated with gefitinib treatment. A few months later, however, she developed recurrent lung tumors. Following the challenging initiation of the TKI erlotinib, she achieved remission without proteinuria. We thus conclude that erlotinib is a potential treatment option in patients with NS associated with gefitinib therapy.

Apurinic/apyrimidinic endonucelase 1 maintains adhesion of endothelial progenitor cells and reduces neointima formation

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein that processes DNA-repair function and controls cellular response to oxidative stress. Endothelial progenitor cells (EPCs) are recruited to oxidative stress-rich injured vascular walls and positively contribute to vascular repair and endothelialization. We hypothesized that APE1 functions for EPCs-mediated inhibition of neointima formation in injured vasculature. EPCs isolated from bone marrow cells of C57BL6 mice (12-16 wk old) were able to survive in the presence of hydrogen peroxide (H2O2; up to 1,000 μM) due to the highly expressed reactive oxygen species (ROS) scavengers. However, adhesion capacity of EPCs was significantly inhibited by H2O2 (100 μM) even though an intracellular ROS was retained at small level. An APE1-selective inhibitor or RNA interference-mediated knockdown of endogenous APE1 in EPCs aggravated the H2O2-mediated inhibition of EPCs-adhesion. In contrast, when APE1 was overexpressed in EPCs using an adenovirus harboring the APE1 gene (APE-EPCs), adhesion was significantly improved during oxidative stress. To examine in vivo effects of APE1 in EPCs, APE-EPCs were transplanted via the tail vein after wire-mediated injury of the mouse femoral artery. The number of adherent EPCs at injured vascular walls and the vascular repair effect of EPCs were enhanced in APE-EPCs compared with control EPCs. Among the cellular functions of EPCs, adhesion is especially sensitive to oxidative stress. APE1 enhances in vivo vascular repair effects of EPCs in part through the maintenance of adhesion properties of EPCs. APE1 may be a novel and useful target gene for effective cellular transplantation therapy.

Diagnosis of IgG4-related systemic disease by cytology of large pericardial effusion with fine needle aspiration

IgG4-related disease has characteristic features as follows; Serum IgG4 is prominently elevated, IgG4-positive plasma cells infiltrate in involved tissues and various mass-forming lesions with fibrosis, such as the autoimmune pancreatitis, salivary gland and retroperitoneum. As this disease comes to attention, the reports of IgG4-related disease have been increasing, but the pathogenesis and exact frequency of the disease remain unknown. We present the case of a 69 year-old male who was admitted to our hospital for evaluation of lower legs edema in July, 2010. He first noticed lower legs edema in 2009 and visited a local clinic, and was diagnosed deep vein thrombosis, received anti-coagulation therapy. However, his edema was getting worth and hydrocele testis was appeared. When he visited our office, he complained of trouble in walking with lower legs edema. On physical examination, his body temperature was 36.2 °C, blood pressure 152/82 mm Hg, and pulse rate 60 per minute. There was no murmur in his cardiac sounds and lungs were clear. On abdominal examination, it was slightly swelling but not any mass were noted. His trunk was edematous, especially in lower legs. The thyroid gland, salivary glands and lymph nodes were not palpable. He had never experienced dry eye and dry mouth. Laboratory findings were as follows: WBC 4390/μL, hemoglobin 10.6 g/dL, platelet 27.3×10/μL, BUN 10 mg/dL, creatinine 1.04 mg/dL, BNP 119.0 pg/mL, CRP b0.10 μg/L. Liver function test was normal. Urinalysis did not demonstrate any protein on dipstick. Rheumatoid factor, anti-nuclear antibody, anti SS-A and anti SS-B, antibodies and antineutrophil cytoplasmic antibody were all negative. IgG, IgA, IgM and IgE were 1769.0 mg/dL, 272.7 mg/dL, 74.8 mg/dL and 276 mg/dL, respectively. Serum level of IgG4 was elevated by 408 mg/dL. A chest radiograph revealed marked cardiomegaly. Echocardiography revealed marked pleural effusion with slightly collapsed right atrium. Computed tomography (CT) revealed also large pericardial effusion (Fig. 1A) and low density area around the aorta (Fig. 1B). The mass, which was suspected retroperitoneal fibrosis (RPF), compressed inferior vena cava and right urinary duct, and induced lower legs edema and right hydronephrosis. After admission, a ureteral stent was placed in order to treat the hydronephrosis. Lip biopsy was done, but there was no significant pathological change. Gallium scintigraphy showed no hot lesions. Autoimmune pancreatitis was ruled out by magnetic resonance pancreatic cholangiopancreatography. Then,we performedpericardiocentesis byfineneedle aspiration on 9 days after admission. The specimen was light yellow, cloudy, TP 5.2 g/dL, albumin 3.0 g/dL, IgG 1604.3 mg/dL, IgG4 451 mg/dL and abundant small sized lymphocytes, rich in IgG4-positive cells (Fig. 2). There was no malignancy. Culture of the pericardial fluid revealed no bacterial infection. Therefore, we diagnosed this case as IgG4-related disease and treated with prednisolone 50 mg/day. Then his edema was rapidly improved. One month after starting steroid therapy, the CT revealed disappearance of pericardial effusion and regression of retroperitoneal mass (Fig. 1C and D). To our knowledge, this is the first case report diagnosed by fine needle aspiration cytology of large pericardial effusion. The pathological characteristic of this disease is rich in IgG4-positive plasma cells with typical fibrosis or sclerosis in the tissue [1]. IgG4related disease sometimes shows retroperitoneal fibrosis (RPF) and caused hydronephrosis as in our case. A biopsy for the retroperitoneal mass is necessary to provide a definitive diagnosis, but is considerably invasive. The major reasons of pericardial effusion are idiopathic pericarditis, infection included tuberculosis and malignancy [2,3]. Secondary to malignancy are frequently observed in lung cancer, breast cancer, and malignant lymphoma. Malignant lymphoma is considered of differential diagnosis in both RPF and pericardial effusion. Fine needle aspiration of pericardial effusion is less invasive compared to retroperitoneal biopsy. Although Sugimoto et al. reported IgG4-related disease caused constrictive pericarditis diagnosed by pericardiectomy [4], pericardial effusion is not a major complication. We showed that the cytology of pericardial effusion was rich in IgG4-positive cells similar to serum, suggesting that IgG4-related disease caused pericardial exudation of the plasma cell. Thus, it is necessary to consider the possibility of IgG4-related disease in the differential diagnosis of pericardial effusion. A pericardiocentesis will be useful to diagnose a IgG4related disease. The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology [5].

Angiotensin II receptor blocker and long-acting calcium channel blocker combination therapy decreases urinary albumin excretion while maintaining glomerular filtration rate

Microalbuminuria is a recognized risk factor and predictor for cardiovascular events in patients with hypertension. We analyzed changes in hypotensive effect, urinary albumin excretion (UAE) and estimated glomerular filtration rate (eGFR) in subjects with hypertension and microalbuminuria as a subanalysis of the results of the Nifedipine and Candesartan Combination (NICE-Combi) Study. A total of 86 subjects with essential hypertension with microalbuminuria (UAE <300 mg g−1 creatinine) were randomly assigned in a double-blind manner to a combination therapy group (standard-dose candesartan at 8 mg per day plus controlled-release (CR) nifedipine 20 mg per day) (n=42) or an up-titrated monotherapy group (candesartan 12 mg per day) (n=44) for 8 weeks of continuous treatment after initially receiving standard-dose candesartan (8 mg per day) monotherapy for 8 weeks (initial treatment). After 8weeks, blood pressure (BP) was significantly reduced in both groups compared with at the end of initial treatment. UAE also showed a significant decrease in the combination therapy group, while there was no significant change of eGFR in either group. A significant positive correlation was seen between BP reduction and UAE after 8 weeks of double-blind treatment in both groups, whereas no significant association was found between ΔUAE and ΔeGFR in either group. These findings show that combination therapy with standard-dose candesartan and nifedipine CR is more effective than up-titrated candesartan monotherapy for reducing BP and improving UAE while maintaining eGFR, and strongly suggest that the combination of an angiotensin II receptor blocker and long-acting calcium channel blocker is beneficial in patients with hypertension and microalbuminuria.

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.