CCN3 inhibits neointimal hyperplasia through modulation of smooth muscle cell growth and migration.

OBJECTIVE: CCN3 belongs to the CCN family, which constitutes multifunctional secreted proteins that act as matrix cellular regulators. We investigated the pathophysiological roles of CCN3 in the vessels. METHODS AND RESULTS: We examined the effects of CCN3 on the proliferation and migration of rat vascular smooth muscle cells (VSMC). CCN3 knockout mice were created, and vascular phenotypes and neointimal hyperplasia induced by photochemically induced thrombosis were investigated. CCN3 suppressed the VSMC proliferation induced by fetal bovine serum. The neutralizing antibody for transforming growth factor-beta did not affect the growth inhibitory effect of CCN3. Moreover, CCN3 enhanced the mRNA expression of cyclin-dependent kinase inhibitors, p21 and p15. Gamma secretase inhibitor, an inhibitor of Notch signaling, partially inhibited the enhanced expression of p21 induced by CCN3. CCN3 also inhibited the VSMC migration. Finally, the histopathologic evaluation of the arteries 21 days after the endothelial injury revealed a 6-fold enhancement of neointimal thickening in the null mice compared with the wild-type mice. CONCLUSIONS: CCN3 suppresses neointimal thickening through the inhibition of VSMC migration and proliferation. Our findings indicate the involvement of CCN3 in vascular homeostasis, especially on injury, and the potential usefulness of this molecule in the modulation of atherosclerotic vascular disease.

Neuropilin-1 in regulation of VEGF-induced activation of p38MAPK and endothelial cell organization.

Vascular endothelial growth factor (VEGF)-A regulates vascular development and angiogenesis. VEGF isoforms differ in ability to bind coreceptors heparan sulfate (HS) and neuropilin-1 (NRP1). We used VEGF-A165 (which binds HS and NRP1), VEGF-A121 (binds neither HS nor NRP1), and parapoxvirus VEGF-E-NZ2 (binds NRP1 but not HS) to investigate the role of NRP1 in organization of endothelial cells into vascular structures. All 3 ligands induced similar level of VEGFR-2 tyrosine phosphorylation in the presence of NRP1. In contrast, sprouting angiogenesis in differentiating embryonic stem cells (embryoid bodies), formation of branching pericyte-embedded vessels in subcutaneous matrigel plugs, and sprouting of intersegmental vessels in developing zebrafish were induced by VEGF-A165 and VEGF-E-NZ2 but not by VEGF-A121. Analyses of recombinant factors with NRP1-binding gain- and loss-of-function properties supported the conclusion that NRP1 is critical for VEGF-induced sprouting and branching of endothelial cells. Signal transduction antibody arrays implicated NRP1 in VEGF-induced activation of p38MAPK. Inclusion of the p38MAPK inhibitor SB203580 in VEGF-A165-containing matrigel plugs led to attenuated angiogenesis and poor association with pericytes. Our data strongly indicate that the ability of VEGF ligands to bind NRP1 influences p38MAPK activation, and formation of functional, pericyte-associated vessels.

Halofuginone prevents extracellular matrix deposition in diabetic nephropathy.

Transforming growth factor-beta (TGF-beta) is known to promote the accumulation of extracellular matrix (ECM) and the development of diabetic nephropathy. Halofuginone, an analog of febrifugine, has been shown to block TGF-beta(1) signaling and subsequent type I collagen production. Here, the inhibitory effect of halofuginone on diabetic nephropathy was examined. Halofuginone suppressed Smad2 phosphorylation induced by TGF-beta(1) in cultured mesangial cells. In addition, the expression of TGF-beta type 2 receptor decreased by halofuginone. Halofuginone showed an inhibitory effect on type I collagen and fibronectin expression promoted by TGF-beta(1). An in vivo experiment using db/db mice confirmed the ability of halofuginone to suppress mesangial expansion and fibronectin overexpression in the kidneys. Moreover, an analysis of urinary 8-OHdG level and dihydroethidium fluorescence revealed that halofuginone reduced oxidative stress in the glomerulus of db/db mice. These data indicate that halofuginone prevents ECM deposition and decreases oxidative stress, thereby suppressing the progression of diabetic nephropathy.

Association of serum levels of antibodies against MMP1, CBX1, and CBX5 with transient ischemic attack and cerebral infarction.

Transient ischemic attack (TIA) is a predictor for cerebral infarction (CI), and early diagnosis of TIA is extremely important for the prevention of CI. We set out to identify novel antibody biomarkers for TIA and CI, and detected matrix metalloproteinase 1 (MMP1), chromobox homolog 1 (CBX1), and chromobox homolog 5 (CBX5) as candidate antigens using serological identification of antigens by recombinant cDNA expression cloning (SEREX) and Western blotting to confirm the presence of serum antibodies against the antigens. Amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) revealed that serum antibody levels were significantly higher in patients with TIA or acute-phase CI (aCI) compared with healthy donors (P < 0.01). Spearman's correlation analysis and multivariate logistic regression analysis demonstrated that levels of anti-MMP1, anti-CBX1, and anti-CBX5 antibodies were associated with age, cigarette-smoking habits, and blood pressure. Thus, serum levels of antibodies against MMP1, CBX1, and CBX5 could potentially serve as useful tools for diagnosing TIA and predicting the onset of aCI.

Targeted disruption of TGF-beta-Smad3 signaling leads to enhanced neointimal hyperplasia with diminished matrix deposition in response to vascular injury.

The role of transforming growth factor (TGF)-beta and its signal in atherogenesis is not fully understood. Here, we examined mice lacking Smad3, a major downstream mediator of TGF-beta, to clarify the precise role of Smad3-dependent signaling in vascular response to injury. Femoral arteries were injured in wild-type and Smad3-null (null) male mice on C57Bl/6 background. Histopathological evaluation of the arteries 1 to 3 weeks after the injury revealed significant enhancement of neointimal hyperplasia in null compared with wild-type mice. Transplantation of null bone marrow to wild-type mice did not enhance neointimal thickening, suggesting that vascular cells in situ play a major role in the response. Null intima contained more proliferating smooth muscle cells (SMC) with less amount of collagen compared with wild-type intima. TGF-beta caused significant inhibition of cellular proliferation in wild-type aortic SMC, whereas the growth of null SMC was only weakly inhibited by TGF-beta in vitro, indicating a crucial role of Smad3 in the growth inhibitory function. On the other hand, Smad3-deficiency did not attenuate chemotaxis of SMC toward TGF-beta. TGF-beta increased transcript level of alpha2 type I collagen and tissue inhibitor of metalloproteinases-1, and suppressed expression and activity of matrix metalloproteinases in wild-type SMC. However, these effects of TGF-beta were diminished in null SMC. Our findings altogether show that the loss of Smad3 pathway causes enhanced neointimal hyperplasia on injury through modulation of growth and matrix regulation in vascular SMC. These results indicate a vasculoprotective role of endogenous Smad3 in response to injury.

Combination of cilostazol and probucol protected podocytes from lipopolysaccharide-induced injury by both anti-inflammatory and anti-oxidative mechanisms.

Podocytes are essential for maintaining kidney glomerular functions. Injuries to podocyte are closely related to the pathological process of proteinuria. However, a treatment for podocyte injury has still not been established. Cilostazol (CSZ) and probucol (PBC) have been shown to possess renoprotective effects. Therefore, we evaluated these drugs in a lipopolysaccharide (LPS)-induced podocyte injury model. 7-week-old female C57BL/6J mice were fed a normal diet or a diet containing 0.3% CSZ, 0.5% PBC, or both for 10 days. Then, mice were intraperitoneally injected with 13 µg g-1 body weight LPS. Both CSZ and PBC decreased LPS-induced albuminuria and co-administration was found to be most effective. These treatments ameliorated the upregulation of monocyte chemoattractant protein 1. In cultured podocytes, CSZ suppressed LPS-induced activation of nuclear factor-kappa B (NF-κB) and phosphorylation of p44/42 mitogen-activated protein kinase (MAPK). PBC reduced LPS-induced activation of NF-κB and reactive oxygen species production. Furthermore, PBC decreased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase4 expression. Our findings suggest that CSZ and PBC are able to inhibit podocyte-injury through different mechanisms, indicating that a combination of these two old drugs is a good treatment option to protect podocytes from injury.

A novel podocyte gene, semaphorin 3G, protects glomerular podocyte from lipopolysaccharide-induced inflammation.

Kidney diseases including diabetic nephropathy have become huge medical problems, although its precise mechanisms are still far from understood. In order to increase our knowledge about the patho-physiology of kidney, we have previously identified >300 kidney glomerulus-enriched transcripts through large-scale sequencing and microarray profiling of the mouse glomerular transcriptome. One of the glomerulus-specific transcripts identified was semaphorin 3G (Sema3G) which belongs to the semaphorin family. The aim of this study was to analyze both the in vivo and in vitro functions of Sema3G in the kidney. Sema3G was expressed in glomerular podocytes. Although Sema3G knockout mice did not show obvious glomerular defects, ultrastructural analyses revealed partially aberrant podocyte foot processes structures. When these mice were injected with lipopolysaccharide to induce acute inflammation or streptozotocin to induce diabetes, the lack of Sema3G resulted in increased albuminuria. The lack of Sema3G in podocytes also enhanced the expression of inflammatory cytokines including chemokine ligand 2 and interleukin 6. On the other hand, the presence of Sema3G attenuated their expression through the inhibition of lipopolysaccharide-induced Toll like receptor 4 signaling. Taken together, our results surmise that the Sema3G protein is secreted by podocytes and protects podocytes from inflammatory kidney diseases and diabetic nephropathy.

Elevated Adiponectin Antibody Levels in Sera of Patients with Atherosclerosis-Related Coronary Artery Disease, Cerebral Infarction and Diabetes Mellitus.

Adiponectin secreted from the adipocytes plays pleiotropic, anti-atherosclerotic roles, such as enhancement of insulin secretion and an increase in energy expenditure. The measurement of levels of circulating adiponectin is useful to evaluate the progression of atherosclerosis-related diseases, such as coronary artery disease (CAD), cerebral infarction (CI) and diabetes mellitus (DM). We examined the serum antibody levels against recombinant adiponectin protein via the amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) method. The results revealed that the antibody levels were significantly higher in patients with CAD, CI and type 2 DM, than in healthy donors. Receiver operating curve analysis showed that the sensitivity was in a range of 41-48% for CAD, CI and DM. Thus, the serum anti-adiponectin antibody levels could be a common marker for atherosclerosis-related diseases.

High glucose-induced upregulation of osteopontin is mediated via Rho/Rho kinase pathway in cultured rat aortic smooth muscle cells.

OBJECTIVE: Osteopontin is upregulated in the diabetic vascular wall and in vascular smooth muscle cells cultured under high glucose concentration. In the present study, we analyzed the mechanism of high glucose-induced upregulation of osteopontin in cultured rat aortic smooth muscle cells. METHODS AND RESULTS: We found that an inhibitor of Rho-associated protein kinase, Y-27632, suppressed osteopontin mRNA expression under high glucose concentration. Transfection of cells with a constitutive active Rho mutant, pSRalpha-myc-RhoDA, enhanced osteopontin mRNA expression. Furthermore, incubation of cells under high glucose concentration activated Rho, indicating that Rho/Rho kinase pathway mediates high-glucose-stimulated osteopontin expression. Treatment of cells with an inhibitor of protein kinase C, GF109203X, and azaserine, an inhibitor of the hexosamine pathway, suppressed high glucose-induced Rho activation. Glucosamine treatment was shown to activate Rho. Treatment of cells with an inhibitor of MEK1, PD98059, suppressed osteopontin mRNA expression under high glucose concentration. Incubation of cells under high glucose concentration activated ERK. Finally, transfection of cells with pSRalpha-myc-RhoDA also activated ERK. CONCLUSIONS: In conclusion, our present findings support a notion that Rho/Rho kinase pathway functions downstream of protein kinase C and the hexosamine pathways and upstream of ERK in mediating high-glucose-induced upregulation of osteopontin expression.

[A case of an elderly type 2 diabetes who had severe diabetic nephropathy without retinopathy].

We encountered a man who developed severe diabetic nephropathy without progression of diabetic retinopathy. He had a 14-year history of diabetes, and had been treated with sulfonylurea, and his HbA1c remained around 6.5%. He was admitted because of systemic edema and dyspnea on effort Laboratory data revealed renal failure and nephrotic syndrome, whereas there was no symptom of diabetic retinopathy. Since diabetic nephropathy usually progresses in parallel with retinopathy, it is atypical to develop severe nephropathy without retinopathy. In this case, longstanding hypertension and his genetic background including angiotensin converting enzyme D/I polymorphism might have played an important role in development of diabetic nephropathy.

Pituitary adenylate cyclase-activating polypeptide protects glomerular podocytes from inflammatory injuries.

Diabetic nephropathy (DN) is a leading cause of end-stage kidney disease; however, there are few treatment options. Inflammation plays a crucial role in the initiation and/or progression of DN. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide, which was originally isolated from the ovine hypothalamus and reportedly has diverse biological functions. It has been reported that PACAP has renoprotective effects in different models of kidney pathology. However, the specific cell types within the kidney that are protected by PACAP have not yet been reported. In this study, we localized VPAC1, one of the PACAP receptors, to glomerular podocytes, which also reportedly has crucial roles not only in glomerular physiology but also in pathology. PACAP was effective in the downregulation of proinflammatory cytokines, such as monocyte chemoattractant protein-1 (MCP-1) and interleukin-6, which had been induced by the activation of toll-like receptor (TLR) with lipopolysaccharide. PACAP also had downregulated the expression of MCP-1 through the protein kinase A signaling pathway; this led to the attenuation of the activation of extracellular signal-regulated kinase and nuclear factor-kappa B signaling. Our results suggested that PACAP could be a possible treatment option for DN through the use of anti-inflammation effects on glomerular podocytes.

Bone marrow-derived vascular cells in response to injury.

Intimal hyperplasia is a key lesion for various vascular disorders such as atherosclerosis, postangioplasty restenosis and transplant arteriopathy. It has widely been accepted that intimal smooth muscle cells (SMC) originate from the medial layer in the same artery. However, recent studies suggest that bone marrow can also provide circulating progenitors for vascular SMC. Bone marrow-derived SMC participate in neointimal formation in animal models of allotransplantation, severe mechanical injury and hyperlipidemia-induced atherosclerosis. In human, transplantation arteriopathy also seems to involve circulating SMC, but their role in atherosclerosis and restenosis remains to be elucidated. Mobilization, differentiation and proliferation steps of SMC progenitors will provide promising targets for novel therapeutic approaches against proliferative vascular diseases.

Identification and characterization of high glucose and glucosamine responsive element in the rat osteopontin promoter.

We have previously reported that high glucose stimulates osteopontin (OPN) expression via a protein kinase C-dependent pathway and a hexosamine pathway in cultured rat aortic smooth muscle cells (SMCs) [Biochem. Biophys. Res. Commun. 258 (1999) 722.]. In the present study, we carried out functional OPN promoter assays using the luciferase expression vector system in cultured rat aortic SMCs to determine a high glucose/glucosamine responsive element. An extensive deletion analysis of the 5'-flanking region of the rat OPN gene revealed that an element involved in high glucose and glucosamine responses was present within a region between -112 and -62 bp of the OPN promoter. This region is highly conserved in the rat, mouse, and human promoters and contains a number of consensus regions, including an E-box and a GC-rich region. Mutation of the E-box or the GC-rich region resulted in a significant loss of both high glucose and glucosamine responses. These results suggest that two cis-acting elements, the E-box and the GC-rich region, are involved at least partly in high glucose/glucosamine-stimulated transcription of the rat OPN gene.

Successful treatment of listerial brain abscess: a case report and literature review.

Listerial brain abscess is extremely rare; only two cases have been reported in Japan. We encountered a female patient with immunoblastic lymphadenopathy, who developed listerial brain abscess after 8 years of treatment with antineoplastic agents and corticosteroids. Brain MRI revealed multiple space occupying lesions, suggesting abscesses which were possibly caused by hematogenous spread of the bacteria. Immediate blood culture enabled early diagnosis, and she entered into complete remission with high-dose ampicillin. Blood culture and brain imaging seem to play a crucial role in making an early diagnosis, and the administration of high dose of antibiotics is recommended for improvement of this disease.

[A report of a case with Werner's syndrome suffering from end-stage renal failure].

A 54-year-old man was found to have hypertension at age 32, and a diagnosis of Werner's Syndrome was made at age 36 when he was examined for hyperlipidemia. Diabetes mellitus was found at age 42. Proteinuria appeared at age 49, and microscopic hematuria was seen at age 50. At age 51, serum creatinin level began to rise and atrophy of bilateral kidneys was observed by abdominal CT. There after, the renal function gradually worsened. At age 53, the serum creatinin level rose to 8.3 mg/dl, and systemic edema as well as loss of appetite appeared, resulting in the initiation of hemodialysis. In Werner's syndrome, though arteriosclerosis arises frequently, case reports with chronic renal failure are extremely rare. To investigate the cause of the renal dysfunction, renal biopsy was performed and the samples were histologically examined, revealing the presence of hypertensive glomerular changes. It is, thus, conceivable that hypertension had played a major role in the progression of renal failure in this case.

Enhanced vascular endothelial growth factor signaling in islets contributes to ß cell injury and consequential diabetes in spontaneously diabetic Torii rats.

AIMS: Spontaneously diabetic Torii (SDT) rats exhibit vascular abnormalities in pancreatic islets as the initial changes at pre-diabetes stage (8 weeks old), which is followed by ß cell deterioration. In the present study, we investigated pathophysiological interactions between ß cells and intra-islet microvasculature of SDT rats at pre- and peri-onset of diabetes. METHODS: SDT rats were treated with Habu snake venom (HSV) to assess its hemorrhagic effects in glomeruli and pancreatic islets. SDT rats were treated with streptozotocin (STZ) to assess acute ß cell fragility toward cytotoxic insult and the late-stage consequence of ß cell ablation in neighboring structures. The receptor tyrosine kinase inhibitor sunitinib was administered to SDT rats to examine its therapeutic effect. RESULTS: HSV administration at 5 weeks old induced severe hemorrhage in and around islets in SDT rats. By contrast, precedent ß cell depletion using STZ ameliorated hemorrhage, inflammation, and fibrosis around the islets at 13 weeks old, which is normally seen in SDT rats of this age. Blockade of vascular endothelial growth factor (VEGF)-like activity attenuated HSV-induced hemorrhage in SDT islets. VEGF release from SDT islets was increased at 13 weeks old but not at 5 weeks old, while interleukin-1ß release was increased as early as 5 weeks old. Sunitinib treatment started at 5 weeks of age inhibited the onset of intra-islet hemorrhage, ß cell loss, and hyperglycemia in SDT rats. CONCLUSIONS: Enhanced VEGF signaling in islets contributes to ß cell injury, microvascular failure, and consequential diabetes in SDT rats.

Atorvastatin ameliorates podocyte injury in patients with type 2 diabetes complicated with dyslipidemia.

We examined the effects of atorvastatin on urinary podocyte excretion. Thirteen patients with type 2 diabetes receiving 2.5mg of rosuvastatin were recruited and the medication was switched to 10mg of atorvastatin for a 24-week period. With the switch to atorvastatin, the urinary excretion of podocytes was significantly reduced.

A low-grade increase of serum pancreatic exocrine enzyme levels by dipeptidyl peptidase-4 inhibitor in patients with type 2 diabetes.

A potential adverse effect of dipeptidyl peptidase-4 inhibitors (DPP-4i) on the pancreas remains controversial. We evaluated the DPP-4i effects on pancreatic amylase and lipase activity in patients with type 2 diabetes. These enzymes were slightly but significantly increased, suggesting DPP-4i cause a low-grade inflammatory change in the exocrine pancreas.

Glucagon-like peptide-1 secretion by direct stimulation of L cells with luminal sugar vs non-nutritive sweetener.

UNLABELLED: Aims/Introduction: Oral ingestion of carbohydrate triggers secretion of glucagon-like peptide (GLP)-1, which inhibits the postprandial rise in blood glucose levels. However, the mechanism of carbohydrate-induced GLP-1 secretion from enteroendocrine L cells remains unclear. In the present study, GLP-1 secretion was examined by meal tolerance tests of healthy Japanese volunteers. MATERIALS AND METHODS: Twenty-one healthy Japanese men participated in the study. The meal tolerance test was performed with modified nutrient compositions, with or without pretreatment with the α-glucosidase inhibitor acarbose, or with substitution of sucrose with an equivalent dose of sweeteners in the meal. Blood concentrations of glucose, insulin, GLP-1, and apolipoprotein (Apo) B-48 were measured. RESULTS: GLP-1 secretion started concomitant with the increase in blood glucose levels 10 min after meal ingestion. Insulin secretion started at 5 min, before the increase in blood glucose levels, reflecting the contribution of direct nutrient stimulation on the former parameter and neural regulation in the latter. Carbohydrate retention in the gut lumen induced by acarbose pretreatment extended postprandial GLP-1 secretion and negated the increase in serum ApoB-48 levels. GLP-1 secretion was markedly decreased by a reduction in the amount of sucrose in the meal and was not restored by an equivalent dose of sweeteners used to compensate for the sweet taste. CONCLUSIONS: The results indicate that direct stimulation of L cells with sugar, but not sweetener, is required for carbohydrate-induced GLP-1 secretion. In addition, inhibition of digestion of dietary carbohydrate by α-glucosidase inhibitors may prevent postprandial hyperglycemia by increasing GLP-1 secretion and by inhibiting glucose absorption. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2011.00163.x, 2011).