Therapeutic Potential of Angiostatin in Diabetic Nephropathy

Sarah X. Zhang, Joshua J. Wang, Kangmo Lu, Robert Mott, Richard Longeras, and Jian-xing Ma Department of Medicine Endocrinology, Department of Cell Biology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma

Angiostatin is a proteolytic fragment of plasminogen and a potent angiogenic inhibitor. Previous studies have shown that angiostatin inhibits retinal neovascularization and reduces retinal vascular permeability in diabetic retinopathy. Here, it is reported for the first time that angiostatin is also implicated in diabetic nephropathy (DN). Angiostatin levels are dramatically decreased in the kidney of streptozotocin-induced diabetic rats. Consistently, diabetic kidneys also showed decreased expression and proteolytic activities of matrix metalloproteinase-2, an enzyme that releases angiostatin from plasminogen. Adenovirus-mediated delivery of angiostatin significantly alleviated albuminuria and attenuated the glomerular hypertrophy in diabetic rats. Moreover, angiostatin treatment downregulated the expression of vascular endothelial growth factor and TGF-␤1, two major pathogenic factors of DN, in diabetic kidneys. In cultured human mesangial cells, angiostatin blocked the overexpression of vascular endothelial growth factor and TGF-␤1 that were induced by high glucose while increasing the levels of pigment epithelium–derived factor, an endogenous inhibitor of DN. Moreover, angiostatin effectively inhibited the high-glucose– and TGF-␤1–induced overproduction of proinflammatory factors and extracellular matrix proteins via blockade of the Smad signaling pathway. These findings suggest that the decrease of angiostatin levels in diabetic kidney may contribute to the pathologic changes such as inflammation and fibrosis in DN. Therefore, angiostatin has therapeutic potential in DN as a result of its anti-inflammatory and antifibrosis activities. J Am Soc Nephrol 17: 475–486, 2006. doi: 10.1681/ASN.2005020217

iabetic nephropathy (DN) is one of the most devastating VEGF is a major angiogenic factor and a vascular permeability microvascular complications of diabetes as well as the factor (13,14). VEGF expression has been shown to increase at D leading cause of ESRD in the United States (1,2). Ap- the early stage of DN in both patients with diabetes and dia- proximately 20 to 40% of the patients with type 1 diabetes and 10 betic animal models (7,15). Blockade of VEGF bioactivity for 6 to 20% of those with type 2 diabetes develop nephropathy (3). The wk abolished glomerular hyperfiltration in streptozotocin earliest pathologic changes of DN are characterized by glomerular (STZ)-induced diabetic rats (16). hypertrophy, the thickening of glomerular basement membrane, The exact mechanisms underlying how these pathologic factors and expanded extracellular matrix (ECM), which lead to glomer- induce nephropathy in diabetes are largely unexplored. Accumu- ular hyperfiltration and microalbuminuria (2,4). Although inten- lating evidence suggests that inflammation plays a crucial role in sified controls of hyperglycemia, BP, and hyperlipidemia reduce DN (17–20). Upregulated expression of proinflammatory cyto- the risks of DN, they do not sufficiently prevent the progression kines, such as TNF-␣, monocyte chemoattractant protein-1 (MCP- from microalbuminuria to overt nephropathy in patients with 1), intercellular adhesion molecule-1 (ICAM-1), and IL-18, is diabetes (1,5,6). closely associated with renal functional damage (21–24). Knockout Growth factors play an important role in the pathogenesis of of ICAM-1 abolished diabetes-induced increase in urinary albu- DN (7). TGF-␤ has been recognized as a major modulator of DN min excretion (UAE), glomerular hypertrophy, and mesangial (8). Overexpression of TGF-␤ in diabetic glomeruli is believed matrix expansion, suggesting that inflammation may be partially to contribute to the ECM accumulation by increasing synthesis responsible for the renal injury in diabetes (19). and decreasing degradation of ECM proteins such as fibronec- Angiostatin, a proteolytic fragment (kringle 1 to 4) of plas- tin and collagen (9–12). Another important growth factor in- minogen, was first identified in the serum and urine of tumor- volved in DN is vascular endothelial growth factor (VEGF). bearing animals (25). Angiostatin is a potent angiogenic inhibitor that specifically inhibits proliferation and induces Received February 25, 2005. Accepted November 25, 2005. apoptosis in vascular endothelial cells (26). In vivo studies have Published online ahead of print. Publication date available at www.jasn.org. shown that angiostatin efficiently arrests tumor growth and metastasis and also suppresses hypoxia-induced retinal neo- S.X.Z. and J.J.W. contributed equally to this study. vascularization (27,28). Recently, we showed that angiostatin Address correspondence to: Dr. Jian-xing Ma, 941 Stanton L. Young Boulevard, BSEB 328B, Oklahoma City, OK 73104. Phone: 405-271-4372; Fax: 405-271-3973; reduces retinal vascular leakage in the STZ-induced diabetic rat E-mail: [email protected] model and oxygen-induced retinopathy model (29). Therapeu-

tic laser photocoagulation increases angiostatin levels in the applied to a precast 10% polyacrylamide gel with 1 mg/ml gelatin. vitreous of patients with diabetic retinopathy (DR) (30). These After renaturation for 1 h and development overnight, the gel was data suggest that decreased expression of angiostatin might be stained with SimplyBlue SafeStain (BioRad) and photographed with associated with the pathogenesis of DR. However, the implica- Imager (Syngene, Cambridge, UK). tion of angiostatin in DN has not been established. In this study, we investigated the function of angiostatin in regulation Intravenous Delivery of Adenovirus-Expressing Angiostatin of TGF-␤ and VEGF in renal cells and measured angiostatin The adenovirus-expressing human angiostatin (Ad-Ang) contains levels in diabetic rat kidney. human plasminogen kringle 1 to 4 under the control of the cytomega- lovirus (CMV) promoter. The adenovirus expressing green fluorescence protein (Ad-GFP) contains a GFP gene under the control of the Materials and Methods CMV promoter. Both of the viral vectors were purchased from Qbio- Animals gene (Montreal, QC, Canada). Diabetic rats were randomly assigned to Brown Norway (BN) rats were purchased from Charles River Labo- three groups 1 wk after the STZ injection. Group 1 received no injection ratories (Wilmington, MA). Care, use, and treatment of all animals in (n ϭ 5), and groups 2 and 3 received an intravenous injection of this study were in strict agreement with the guidelines set forth by the Ad-Ang (n ϭ 7) and Ad-GFP as controls (n ϭ 5), respectively, at a dose University of Oklahoma. of 4 ϫ 1010 viral particles per rat.

Induction of Experimental Diabetes Diabetes was induced by an intraperitoneal injection of STZ (Sigma, Evaluation of Rat Microalbuminuria The 24-h urine collected from each rat was centrifuged at 2000 ϫ g for St. Louis, MO) at 50 mg/kg body wt into 8-wk-old BN rats after an 5 min. The urine creatinine levels were determined using the Quan- overnight fasting. Blood glucose levels were measured at 48 h after STZ tiChrom Creatinine Assay Kit (BioAssay Systems, Hayward, CA), fol- injection. The animals with blood glucose Ͼ350 mg/dl were used as lowing the manufacturer’s protocol. The concentration of urine albu- diabetic rats. min was measured by ELISA (Bethyl Laboratories Inc., Montgomery, TX). The UAE was normalized by creatinine excretion and expressed as Cell Culture mg albumin/mg creatinine in 24-h urine. Primary human glomerular mesangial cell (HMC) culture was de- scribed previously (31). Cells of passages 6 to 10 were used in the experiments. An immortalized mouse podocyte cell line was a gift from Quantification of Glomerular Volume and Glomerular Dr. Peter Mundel (Albert Einstein College of Medicine, Bronx, NY) and Cell Numbers maintained as documented previously (32). After reaching 80% conflu- The kidneys were fixed in 4% formaldehyde solution and paraffin- ␮ ence, cells were exposed to 0.5% FBS for 12 h followed by the treatment embedded, and 4- m sections were cut. The sections were stained with with desired reagents. Masson’s trichrome staining and read by two observers who were unaware of experimental protocol under a microscope (33). The glo- Western Blot Analysis of Angiostatin, TGF-␤1, VEGF, merular areas were measured using SPOT Advanced Software (Diag- and ICAM-1 nostic Instruments, Inc., Sterling Heights, MI) and averaged from 120 to 150 glomeruli per kidney (34). The glomerular volume was calculated The kidney tissue was homogenized and centrifuged at 4°C. The by the formula V ϭ area1.5ϫ 1.38/1.1 (35). The cell numbers in protein concentration in the supernatant was measured with the Bio- G glomeruli were counted, and the average glomerular cell number was Rad DC protein assay (BioRad Laboratories, Hercules, CA). Fifty mi- obtained from 100 glomeruli per kidney. crograms of protein from each sample was blotted by an anti-angiostatin or anti–TGF-␤1 antibody (R&D Systems, Minneapolis, MN). The same membranes were stripped and reblotted by anti-VEGF and anti– Measurement of VEGF, TGF-␤1, Pigment Epithelium– ICAM-1 (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies. Derived Factor, MCP-1, and Fibronectin Protein Level by ELISA Determination of Matrix Metalloproteinase-2 mRNA Levels The protein levels of VEGF, TGF-␤1, pigment epithelium–derived by Real-Time Reverse Transcription–PCR factor (PEDF), and fibronectin in the cell culture medium or in the Primers specific for matrix metalloproteinase-2 (MMP-2; forward kidney tissue homogenate were quantified using the commercial Quan- ␤ 5Ј-ggccaactacaacttcttcc-3Ј, reverse 5Ј-ccatcatggattcgagaaaa-3Ј) were tikine VEGF or TGF- 1 ELISA Kit (R&D Systems, Minneapolis, MN), used for real-time reverse transcription–PCR (RT-PCR). The PCR was PEDF ELISA kit (Chemicon Inc., Temecula, CA), and fibronectin ELISA performed using GeneAmp RNA PCR kit and SYBR Green PCR Master (Assaypro, Winfield, MO), respectively, according to the manufactur- Mix (Applied Biosystems, Foster City, CA). The efficiency of real-time er’s protocols. The amount of MCP-1 was measured by a MCP-1 ELISA kit (Chemicon Inc.). PCR is 99.1%. The average threshold cycle (CT) of fluorescence units was used to analyze the mRNA levels. The MMP-2 mRNA levels were normalized by 18s ribosomal RNA levels. Quantification was calcu- ⌬ ⌬ Smad Nuclear Translocation Assay ϭ ( CT) lated as follows: mRNA levels (percent of control) 2 with Primary HMC were cultured on four-chamber slides (Nalge Nunc ⌬ ϭ Ϫ ⌬ ⌬ ϭ⌬ Ϫ CT CT, MMP-2 CT, 18S RNA and ( CT) CT, normal sample International Corp., Naperville, IL) to reach 80% confluence. After ⌬ CT, STZ-diabetic sample. exposure to 2.5 ng/ml TGF-␤1 with or without 100 nM angiostatin for 1 h, the cells were fixed immediately with 4% paraformaldehyde. The MMP-2 Activity Assay by Gelatin Zymography cells were incubated with anti–Smad2/3 antibody (1:200; Upstate USA, Gelatinolytic activity of MMP-2 was analyzed by gelatin zymogra- Inc., Lake Placid, NY) for 2 h and then incubated with cy3-conjugated phy (BioRad Laboratories) following the manufacturer’s protocol. Fif- donkey anti-rabbit antibody for 1 h. The slide was visualized under a teen micrograms of tissue extracts or 20 ␮L of culture medium was fluorescence microscope (Olympus, Hamburg, Germany). J Am Soc Nephrol 17: 475–486, 2006 Angiostatin in Diabetic Nephropathy 477

Statistical Analyses 1A). In the retina, there were only low levels of plasminogen Statistical analysis used t test. Statistical difference was considered but not its proteolytic fragments (Figure 1A). Ͻ significant at P 0.05. Angiostatin levels were determined in the kidney from BN rats with diabetes for 6 wk, which had developed polyuria and mi- Results croalbuminuria (data not shown). The results showed that angiosta- Angiostatin Is Generated in Normal Rat Kidney and tin levels were drastically decreased in diabetic kidney (Figure 1, B Decreased in Diabetic Kidney and C). In contrast, the same gel showed that the intact plasminogen Western blot analysis showed that high levels of plasmino- levels were significantly higher in diabetic kidney than that in normal gen and its proteolytic fragments exist in the kidney and liver control, demonstrating a decreased proteolysis of plasminogen lead- (Figure 1A). Two forms of angiostatin with apparent molecular ing to a reduced release of angiostatin (Figure 1, B and C). In the same weights of approximately 50 and 38 kD were identified in the samples, however, TGF-␤1 levels were significantly increased in the kidney, but only the 38-kD form was found in the liver (Figure diabetic kidneys (Figure 1D).

Figure 1. Western blot analysis of angiostatin in the kidneys of normal and diabetic rats. (A) Western blot analysis of angiostatin in the kidney, liver, and retina of normal adult rats. Note all of the panels in A are from the same gel under different exposures. (B) Western blot analysis of angiostatin in the kidney from rats with diabetes for 6 wk and age-matched normal controls. Equal amounts (50 ␮g) of total protein from each sample were blotted with a specific anti-angiostatin antibody, which recognizes angiostatin as well as its precursor, protein plasminogen and plasmin. (C) Densitometry of Western blot. The results showed that angiostatin existed in two forms with apparent molecular weights of 50 and 38 kD in the normal rat kidney and dramatically decreased in the diabetic kidney. (D) Western blot analysis of TGF-␤1 in the same kidneys as those used in B. The membrane was stripped and reblotted with an anti–␤-actin antibody. 478 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 475–486, 2006

Decreased MMP-2 Expression and Angiostatin Generation levels in kidneys from diabetic and control rats. The results in Diabetic Cortex and Medulla showed that angiostatin levels were significantly decreased The protein levels of angiostatin in diabetic cortex and in both diabetic cortex and medulla, when compared with medulla were determined by Western blot analysis. As the that in normal controls (Figure 2A). In the same samples, increase of ICAM-1 and VEGF levels has been shown to ICAM-1 and VEGF levels showed substantial increases over correlate closely with the early abnormalities in diabetic the normal levels (Figure 2A), suggesting that the decrease of kidney (7,36,37), we also determined the ICAM-1 and VEGF angiostatin is correlated with the increase of angiogenic and

Figure 2. Decreased angiostatin generation and matrix metalloproteinase-2 (MMP-2) expression in the cortex and the medulla of diabetic kidney. (A) Western blot analysis of angiostatin, intercellular adhesion molecule-1 (ICAM-1), and vascular endothelial growth factor (VEGF) in the renal cortex and the medulla from Brown Norway (BN) rats with 6 wk of diabetes and age-matched normal controls. Equal amounts (50 ␮g) of total protein from each sample were blotted with an anti-angiostatin antibody. The same membrane was stripped and reblotted sequentially with antibodies specific to rat ICAM-1 and VEGF. Bottom panels show the densitometry of the specific bands in Western blots above. (B) Quantification of the MMP-2 mRNA in the cortex and the medulla by real-time reverse transcription–PCR and normalized by 18s RNA levels. The average mRNA level was expressed as percentage of respective controls (mean Ϯ SD, n ϭ 4). (C) MMP-2 activity analyzed by zymography. Fifteen micrograms of kidney extracts was loaded onto a precast 10% polyacrylamide gel that was co-polymerized with 1 mg/ml gelatin. After electrophoresis, the gel was renatured and developed. A clear band was observed at 66 kD, demonstrating the area digested by active MMP-2. (D) MMP-2 activity in the conditioned medium from human mesangial cells (HMC) and podocytes after a 48-h culture. (E) MMP-2 activity in cultured HMC. After incubation with high glucose (30 mM) for 72 h or TGF-␤1 (5 ng/ml) for 48 h, the MMP-2 activity in the conditioned medium was determined by zymography. (F) MMP-2 activity in cultured podocytes that were treated with high glucose (30 mM) or mannitol as osmotic control for 72 h. C through F are reversed images of zymographs. J Am Soc Nephrol 17: 475–486, 2006 Angiostatin in Diabetic Nephropathy 479 proinflammatory factors, which both may play roles in the diabetic kidneys. The results showed that at 4 wk after the onset pathogenesis of DN. of diabetes, the glomerular volume was increased by 30% in To explore further the possible mechanism that is responsible diabetic rats when compared with age-matched nondiabetic for the decrease of angiostatin in diabetic kidney, we investi- controls (P Ͻ 0.01, n ϭ 5; Figure 3B). Angiostatin treatment gated the expression of MMP-2, which was recognized as a successfully attenuated the glomerular hypertrophy (P Ͻ 0.01, major protease responsible for the release of angiostatin from n ϭ 5; Figure 3B). We also counted the glomerular cell numbers plasminogen (38). Real-time RT-PCR demonstrated that mRNA in experimental and control groups. The glomerular cell num- levels of MMP-2 were drastically decreased in both the cortex bers, however, did not show any significant difference between and the medulla from rats with 6 wk of diabetes (Figure 2B). the diabetic kidneys and normal control (data not shown). The gelatin zymography showed that enzymatic activities of There was also no difference in cell numbers between the MMP-2 were also significantly lower in diabetic cortex and Ad-Ang–treated group and control virus–treated groups (data medulla (Figure 2C). These results suggest that the decreased not shown). expression of MMP-2 might be responsible, at least in part, for As TGF-␤ is a major pathogenic factor responsible for the the decreased angiostatin levels in diabetic kidney. glomerular hypertrophy in the kidney in diabetic rat models To identify the cause of the MMP-2 decrease in diabetic (9,12,32), we next examined the effect of Ad-Ang on TGF-␤1 kidney, we examined the effects of high glucose and TGF-␤1on expression in diabetic kidneys. The results showed that the MMP-2 expression in cultured glomerular cells. As endothelial renal TGF-␤1 levels were significantly increased in the diabetic cells in the glomeruli express only very low levels of MMP, we kidney with or without the Ad-GFP virus treatment. Ad-Ang first compared the MMP-2 activity in the cultured HMC and delivery significantly decreased TGF-␤ levels in diabetic kid- the podocyte cell line (39). The results showed that the MMP-2 neys (P Ͻ 0.05, n ϭ 5; Figure 3C). activity in the conditioned medium from HMC was 10-fold In addition, we determined the effect of Ad-Ang on VEGF higher than that from podocytes after a 48-h culture (P Ͻ 0.01; levels in diabetic rat kidneys, as VEGF is another known patho- Figure 2D), consistent with the previous study showing the genic factor in DN. The results showed that at 3 wk after the high expression of MMP-2 in mesangial cells but not in podo- onset of diabetes, the renal VEGF levels were significantly cyte or endothelial cells (40). In HMC that were treated with 30 elevated, consistent with the increase of TGF-␤1 levels and mM glucose for 72 h, MMP-2 activities in the conditioned glomerular hypertrophy. The Ad-Ang treatment significantly medium were significantly lower than those in the normal reduced the VEGF levels in the kidney of diabetic rats (P Ͻ 0.05, control (5 mM glucose) or mannitol osmotic control (5 mM n ϭ 5; Figure 3D). glucose ϩ 25 mM mannitol; Figure 2E). TGF-␤1 (5 ng/ml) treatment for 48 h also significantly decreased the MMP-2 Angiostatin Inhibits High-Glucose–Induced Overexpression secretion from HMC (Figure 2E). The decrease of MMP-2 ac- of VEGF and TGF-␤ in HMC tivity that was induced by high glucose was also observed in We determined the direct effect of angiostatin on VEGF expres- podocytes (Figure 2F). These results suggest that both hyper- sion in cultured glomerular cells. After incubation of HMC with a glycemia and overexpression of TGF-␤ contribute to the de- high-glucose (30 mM) medium for 48 h, secreted VEGF levels crease of angiostatin in diabetic kidney. were significantly increased over the mannitol control (Figure 4A). Angiostatin decreased high-glucose–induced VEGF secretion to Decrease of Albuminuria and Renal VEGF and TGF-␤ the level of the mannitol control. This angiostatin effect was also Levels and Attenuation of Glomerular Hypertrophy in observed at 72 and 96 h of the treatment (Figure 4A). Diabetic Rats Treated with Ad-Ang The direct effect of angiostatin on TGF-␤1 secretion from HMC Ad-Ang and the same amount of the control virus, Ad-GFP, was also evaluated in cultured HMC. After 48 h of incubation with were injected separately into the diabetic rats 1 wk after the high glucose in the absence or presence of angiostatin, TGF-␤1 levels onset of diabetes. The UAE was evaluated at 2 wk after the in the culture medium were comparable to that in the cells that were adenovirus delivery. The results showed that in 3-wk diabetic exposed to mannitol for osmotic controls (Figure 4B). After 72- and rats without virus injection, the UAE was increased by 30-fold 96-h incubations, the TGF-␤1 secretion was significantly increased by over that in age-matched nondiabetic controls (P Ͻ 0.01, n ϭ 5; high glucose over those in the mannitol control (Figure 4B). Angiosta- Figure 3A). No significant difference in the UAE was observed tin significantly inhibited high-glucose–induced TGF-␤1 secretion at between the diabetic rats that received Ad-GFP injection or not, 72 and 96 h of the treatment (Figure 4B). indicating that the injection of adenovirus vector did not affect albuminuria. The UAE in the rats that received Ad-Ang injec- Angiostatin Decreases High-Glucose– and TGF-␤1–Induced tion was significantly lower than that in the rats that received MCP-1 Secretion in HMC control virus injection and in untreated diabetic rats (P Ͻ 0.01, MCP-1 is one of the most important proinflammatory che- n ϭ 5; Figure 3A), suggesting that angiostatin protects the mokines implicated in the pathogenesis of DN (17,43,44). High kidney from early diabetic injury. glucose is known to upregulate MCP-1 expression via the Glomerular hypertrophy has been well characterized in the NF-␬B activation in cultured renal mesangial cells (45). In our early STZ-induced diabetes and is closely linked to glomerular study, we examined the effects of angiostatin on MCP-1 secre- hyperfiltration and microalbuminuria in rats (34,41,42). We tion that was induced by high glucose and TGF-␤1. After evaluated the effect of angiostatin on glomerular volume in incubation with high glucose for 48 h, the MCP-1 secretion was 480 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 475–486, 2006

Figure 3. Effect of angiostatin on microalbuminuria, glomerular hypertrophy, and overproduction of renal VEGF and TGF-␤ in diabetic rats. One week after the onset of diabetes, the diabetic rats received an intravenous injection of adenovirus-expressing human angiostatin (Ad-Ang) or the same titer of Ad-GFP as the control. (A) Two weeks after the virus injection, 24-h urine was collected from each rat to measure the albumin and creatinine. Total amounts of albumin in the urine were normalized by creatinine concentrations (mean Ϯ SD, n ϭ 5). (B) The glomerular volume was measured in the kidneys at 3 wk after the Ad-Ang delivery (mean Ϯ SD, n ϭ 5). An average of 120 to 150 glomeruli were measured in each kidney. The TGF-␤1 (C) and VEGF (D) levels in the kidney were measured by ELISA in the rats at 3 wk after the virus delivery and normalized by total protein concentrations. The results showed that renal VEGF and TGF-␤1 levels in the rats that received Ad-Ang injection were significantly lower than that in the rats that received Ad-GFP injection (P Ͻ 0.05, n ϭ 3). increased by 2.4-fold, when compared with that of the mannitol control cells (P Ͻ 0.001, n ϭ 4; Figure 6A). Angiostatin effi- control (Figure 5A). Angiostatin from 2 to 250 nM decreased ciently inhibited the TGF-␤1–induced PEDF decrease in a con- MCP-1 secretion in a concentration-dependent manner. centration-dependent manner (Figure 6A). Moreover, under Similarly, TGF-␤1 (5 ng/ml) treatment for 48 h significantly normal conditions, 50 nM angiostatin significantly increased increased the MCP-1 secretion in HMC (Figure 5B). Angiostatin PEDF secretion, suggesting that angiostatin may be a potential (2 to 50 nM) blocked TGF-␤1–induced MCP-1 increase in a positive regulator of PEDF secretion (Figure 6A). concentration-dependent manner, suggesting an anti-inflam- Angiotensin II has been shown as a crucial factor inducing matory effect of angiostatin. glomerulosclerosis in DN through stimulating ECM production and inhibiting ECM degradation (49). Our study showed that Angiostatin Prevents High-Glucose–Induced 50 ng/ml angiotensin II decreased PEDF secretion in HMC. Downregulation of PEDF in Cultured HMC Angiostatin (2 to 250 nM) effectively prevented the PEDF de- PEDF is an endogenous angiogenic inhibitor that has been crease induced by angiotensin II (Figure 6B). implicated in DR (46–48). Recently, we reported that PEDF acts as an endogenous inhibitor of TGF-␤ and VEGF in the kidney Angiostatin Blocks High-Glucose– and Angiotensin II– (31). In our study, we determined the effect of angiostatin on Induced Fibronectin Secretion from HMC PEDF secretion in cultured HMC that were insulted by TGF-␤1 In DN, the overproduction of ECM proteins, such as fi- and angiotensin II, two common pathologic factors of DN bronectin and collagen, is a major causative factor that is re- (9,49). sponsible for the glomerular hyperfiltration and mesangial ex- After incubation with 5 ng/ml TGF-␤1 for 48 h, PEDF secre- pansion in diabetic kidneys (50). In cultured primary HMC, an tion was decreased by three-fold compared with that in the exposure to high glucose (30 mM) for 48 h led to significant J Am Soc Nephrol 17: 475–486, 2006 Angiostatin in Diabetic Nephropathy 481

Figure 5. Inhibitory effect of angiostatin on monocyte chemoattractant protein-1 (MCP-1) secretion in HMC. HMC were incu- Figure 4. Angiostatin-induced downregulation of VEGF and bated with 5 ng/ml TGF-␤1 (A) or 30 mM glucose (B) in the TGF-␤ expression in HMC that were cultured in high glucose. absence or presence of different concentrations of angiostatin HMC were incubated with high glucose (30 mM) in the absence (0.4 to 250 nM) for 48 h. MCP-1 levels in the medium were or presence of 100 nM angiostatin; 5 mM glucose ϩ 25 mM measured by ELISA. The results were normalized by total mannitol was used as the osmotic control. The medium was protein concentrations in the medium and expressed as pg/mg harvested at 48, 72, and 96 h after the incubation. The VEGF of total protein (mean Ϯ SD, n ϭ 3). Values that are statistically level (A) and TGF-␤1 level (B) in the medium were measured different from the normal glucose controls are indicated by by ELISA. The results were normalized by total protein con- **P Ͻ 0.01. Values that are statistically different from the cells centration in the medium and expressed as pg/mg total protein that were treated with high glucose or TGF-␤1 are indicated by (mean Ϯ SD, n ϭ 3). Values that are statistically different from †P Ͻ 0.05 or ‡P Ͻ 0.01 the normal controls are indicated by *P Ͻ 0.05 or **P Ͻ 0.01. Values that are statistically different from the high glucose the effect of TGF-␤1 was abolished in an angiostatin concentra- † Ͻ ‡ Ͻ alone are indicated by P 0.05 or P 0.01. tion-dependent manner (Figure 8A). To exclude the possible contamination of fibronectin from the serum in the culture medium, we confirmed by Western blot analysis using an increases of fibronectin secretion (Figure 7A). At concentrations antibody specific for cellular fibronectin the results of fibronec- from 10 to 250 nM, angiostatin decreased the fibronectin secre- tin changes that were obtained from ELISA (data not shown). tion in a concentration-dependent manner (Figure 7A). In the To explore further the possible mechanism underlying the ␤ cells that were exposed to 50 ng/ml angiotensin II for 48 h, the effect of angiostatin on TGF- 1–induced fibronectin produc- fibronectin production was dramatically increased (Figure 7B). tion, we determined whether the inhibitory effect of angiostatin ␤ Angiostatin (10 to 250 nM) showed a concentration-dependent on TGF- is through blocking Smad activation, which is a major ␤ inhibition of fibronectin secretion induced by angiotensin II signaling pathway mediating TGF- functions. HMC were in- ␤ (Figure 7B). cubated with TGF- 1 (5 ng/ml) in the absence or presence of 100 nM angiostatin for 1 h followed by immunocytochemistry Angiostatin Suppresses TGF-␤1–Induced Fibronectin assay with an anti-Smad2/3 antibody. The results showed that Production via Blocking Smad2/3 Activation in HMC when compared with control cells (Figure 8B-a), TGF-␤1 stim- As a crucial mediator of ECM production and accumulation, ulated Smad2/3 expression and translocation from the cyto- TGF-␤ strongly stimulated HMC to produce fibronectin. After plasm to the nuclei, a critical step in TGF-␤ function (Figure incubation with TGF-␤1 for 48 h, the fibronectin secretion from 8B-b). Treatment with 100 nM angiostatin significantly blocked HMC was increased by four-fold over the control. In the pres- the nuclear translocation of Smad2/3 induced by TGF-␤1 (Fig- ence of different concentrations of angiostatin (0.4 to 250 nM), ure 8B-c). 482 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 475–486, 2006

Figure 6. Angiostatin-induced upregulation of pigment epithe- Figure 7. Angiostatin blocks high-glucose–induced fibronectin lium–derived factor (PEDF) expression in HMC. Cultured secretion from HMC. HMC were incubated with 30 mM glu- ␤ HMC were challenged with 5 ng/ml TGF- 1 (A) or 50 ng/ml cose (A) or 50 ng/ml of angiotensin II (B) in the absence or angiotensin II (B) in the absence or presence of different doses presence of different concentrations of angiostatin (0.4 to 250 of angiostatin (0.4 to 250 nM) for 48 h. PEDF levels in the nM) for 48 h. Fibronectin that was secreted into the medium medium were measured by ELISA. The results were normal- was measured by ELISA. The fibronectin level in the serum ized by total protein concentrations in the medium and ex- (0.5%) was measured and subtracted. The results were normal- Ϯ ϭ pressed as ng/mg total protein (mean SD, n 3). Values that ized by total protein concentrations in the medium and ex- are statistically different from the normal controls are indicated pressed as ␮g/mg total protein (mean Ϯ SD, n ϭ 3). Values that Ͻ Ͻ by *P 0.05 or **P 0.01. Values that are statistically different are statistically different from the normal controls are indicated ␤ † Ͻ from TGF- – or angiotensin II–treated are indicated by P by **P Ͻ 0.01. Values that are statistically different from cells ‡ Ͻ 0.05 or P 0.01. that were exposed to high glucose or angiotensin II are indicated by ‡P Ͻ 0.01.

Angiostatin Does not Affect Growth of HMC To determine whether the inhibitory effect of angiostatin on the diabetic kidney may contribute to pathologic changes of fibronectin production is through affecting the proliferation of DN. This study for the first time reveals the implication of mesangial cells, we examined the effect of angiostatin on HMC angiostatin in DN. growth. The results showed that angiostatin did not affect the Angiostatin was first identified as internal fragments of plas- HMC viability under either high-glucose condition (30 mM) or minogen in the serum and urine of tumor-bearing animals (25). normal-glucose condition (5 mM; Figure 9), suggesting that the Although angiostatin was given a single name, in fact angiosta- angiostatin-induced decreases of fibronectin and TGF-␤1 levels tin refers to several fragments of plasminogen, such as kringle are not a result of reduced cell numbers. 1 to 3, kringle 1 to 4, kringle 1 to 4.5, and kringle 1 to 5, which all showed anti-angiogenic activities (51). In the kidney homog- Discussion enate from normal BN rats, we observed two forms of angiosta- Angiostatin is known as an inhibitor of angiogenesis, tumor tin at molecular weights of approximately 50 and 38 kD, con- growth, and metastasis (25,28,51,52). This study identified a sistent with the two forms of angiostatin reported by Basile et al. novel function of angiostatin: Inhibiting high-glucose–induced (53) recently. Only the 38-kD form was observed in the liver. overexpression of VEGF and TGF-␤1 and also suppressing No angiostatin or other proteolytic fragments of plasminogen inflammation and fibrosis in kidney cells under diabetic were observed in the retina, likely because of the low abun- stresses. Moreover, we demonstrate that generation of endog- dance of plasminogen in the retina. These results suggest that enous angiostatin is significantly decreased in the kidney of a the generation of angiostatin is tissue specific. diabetic rat model, and delivery of angiostatin significantly Previous studies have shown that angiostatin, as an endog- alleviates functional abnormalities in the kidney of diabetic enous angiogenic inhibitor, is implicated in DR (29). In this rats. These findings suggest that angiostatin may serve as an study, we demonstrated that angiostatin levels were decreased endogenous inhibitor of inflammation and fibrosis in the nor- in the kidney of rats with 6 wk of diabetes, which are known to mal kidney, and the decrease of the angiostatin generation in have functional and structural abnormalities of DN, including J Am Soc Nephrol 17: 475–486, 2006 Angiostatin in Diabetic Nephropathy 483

Figure 8. Angiostatin blocks TGF-␤1 function via blockade of Smad2/3 activation in HMC. (A) HMC were treated with 5 ng/ml TGF-␤1 in the absence or presence of different concentrations of angiostatin (0.4 to 250 nM) for 48 h. Fibronectin that was secreted into the medium was measured by ELISA, normalized by total protein concentrations in the medium, and expressed as ␮g/mg of total protein (mean Ϯ SD, n ϭ 3). Values that are statistically different from the normal controls are indicated by **P Ͻ 0.01. Values that are statistically different from cells that were treated with TGF-␤1 without angiostatin are indicated by †P Ͻ 0.05 or ‡P Ͻ 0.01. (B) HMC were incubated with 5 ng/ml TGF-␤ in the absence or presence of 100 nM angiostatin for 1 h. The cells were fixed and stained by an anti-Smad2/3 antibody and visualized under a fluorescence microscope. Significant increase of Smad2/3 expression and nuclear translocation were observed in the cells that were exposed to TGF-␤1 (B-b), when compared with that in the control cells without TGF-␤1 treatment (B-a). (B-c) Angiostatin (100 nM) effectively blocked the TGF-␤1–induced upregulation and translocation of Smad2/3. Magnification, ϫ400 in B.

polyuria, microalbuminuria, and renal inflammation (16). This mRNA levels in the podocytes and endothelial cells were only result for the first time suggests a potential role of decreased 33 and 18%, respectively, of that in the mesangial cells (40). Our angiostatin levels in the development or progression of DN. study showed that the MMP-2 activity in conditioned medium Moreover, our results demonstrate that plasminogen levels, in from HMC culture was 10-fold higher than that from mouse contrast to the decreased angiostatin levels, are significantly podocytes, suggesting a predominant role of mesangial cells in higher in the diabetic kidney, suggesting that the proteolytic MMP-2 production. Moreover, our studies showed that expo- release of angiostatin from plasminogen, rather than the ex- sure of the cells to high glucose or TGF-␤ increased the MMP-2 pression of the plasminogen gene, is deficient in diabetes. This activity in both mesangial cells and podocytes, suggesting that conclusion is further supported by the observation that both the the decrease of MMP-2 expression and subsequent decrease of expression and the activity of MMP-2 are suppressed in dia- angiostatin levels in the diabetic kidney could be induced by betic kidney, as MMP-2 has been shown to release angiostatin hyperglycemia and the increase of TGF-␤ levels in DN. from plasminogen (38). To investigate the function of angiostatin in the kidney, we Although endothelial cells in the retina are recognized as a delivered recombinant angiostatin via an adenovirus-mediated major source of MMP, previous studies showed that MMP are gene. The angiostatin gene delivery indeed reduced UAE in expressed mainly in mesangial cells and podocytes in the glo- diabetic rats almost to the normal level, whereas the control meruli (39,40). In the three types of glomerular cells, MMP-2 adenovirus that expressed GFP did not affect the microalbu- 484 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 475–486, 2006

tribute to the overexpression of these two pathogenic factors of DN. PEDF is recognized as an anti-angiogenic factor and neuro- trophic factor (47). Recently, we reported that PEDF is expressed at high levels in the kidney and has a protective effect against DN (31). In our study, we also determined the effects of angiostatin on PEDF expression in kidney cells under diabetic insults. The results showed that angiostatin at low doses prevented the PEDF decrease that was induced by TGF-␤ and angiotensin II, suggesting that angiostatin enhances the production of endogenous protective factors under diabetic stresses. The mechanism underlying the upregulation of PEDF by angiostatin is to be elucidated further. Accumulating evidence has suggested that chronic inflammation is a major contributor to DN (17–20). In the early stage of DN, several proinflammatory factors such as MCP-1, TNF-␣, ICAM-1, and IL-18, have been found to be upregulated (21–23). MCP-1 is a major chemokine inducing monocyte migration and differentiation to macrophages, which augment ECM production and interstitial fibrosis in diabetic kidney (17,43,44). In this study, we demonstrate that angiostatin significantly blocks high-glucose– and TGF-␤–induced MCP-1 secretion in mesangial cells, suggesting that angiostatin inhibits inflammation in DN. These results were consistent with the recent report about Figure 9. Angiostatin had no effect on cell proliferation in HMC. The MTT assay was used to determine the viable HMC number the anti-inflammatory effect of angiostatin (56). after treatments with different concentrations of angiostatin for Overproduction of ECM proteins and mesangial matrix ex- 3 d under both normal-glucose (A) and high-glucose (B) con- pansion are the early characteristics of DN that contribute to ditions. The results showed that angiostatin had no effect on microalbuminuria (1,57). As mesangial cells are the major pro- viable cell numbers of HMC. ducer of ECM, we used primary HMC as a model to determine whether angiostatin could block the ECM protein secretion that is induced by different diabetic stressors, including high glu- minuria under the same conditions, suggesting a potent effect cose concentration, angiotensin II, and TGF-␤. The results of angiostatin on the inhibition of microalbuminuria. As mi- showed that angiostatin blocked the fibronectin overproduc- croalbuminuria has been shown to be closely linked with glo- tion that is induced by all of these stressors. At the same merular hypertrophy in the early stage of DN, we further concentration, however, angiostatin had no effect on mesangial determined the effect of angiostatin on glomerular hypertrophy cell growth, suggesting that the inhibition of fibronectin pro- (34,41,42,54,55). Consistent with the reduction of UAE, the glomerular hypertrophy was significantly attenuated in angiosta- duction is not a result of changed viable cell numbers. This tin-treated rats. Furthermore, we investigated the effect of an- result is consistent with our in vivo studies showing that the giostatin on the expression of VEGF and TGF-␤, which are adenovirus-delivered angiostatin treatment ameliorated hyper- recognized as the major pathogenic factors responsible for the trophy of glomeruli but not the hyperplasia in the diabetic glomerular hypertrophy and proteinuria (15,34). The results kidneys. showed that the kidney VEGF and TGF-␤ levels were signifi- Most previous studies of angiostatin focused primarily on its cantly decreased in angiostatin-treated rats, suggesting that therapeutic potential in tumor and retinal neovascularization angiostatin is a potent inhibitor of VEGF and TGF-␤ expression (51,52,58,59). The function of angiostatin in the kidney has not in the kidney. been studied previously. Our results showed that angiostatin ␤ The potent effect of angiostatin on the inhibition of VEGF and blocks the expression and function of VEGF and TGF- but TGF-␤ in diabetic kidney was confirmed further by in vitro enhances the expression of endogenous protective factor PEDF. studies. In cultured HMC, angiostatin efficiently blocked high- It also inhibits inflammation and ECM production under dia- glucose–induced overexpression of TGF-␤ and VEGF. More- betic stresses. Therefore, the decreased generation of angiosta- over, angiostatin inhibited the function of TGF-␤, i.e., blocking tin in diabetic kidney may contribute to the pathologic changes TGF-␤–induced MCP-1 and fibronectin expression. This effect of DN. Although our study suggests that ATP synthase is a is at least partially mediated by the inhibition of Smad activa- possible receptor for angiostatin in mesangial cells (data not tion, a major signaling pathway that mediates TGF-␤ activities. shown), the mechanisms that are responsible for the angiostatin These results further suggest that angiostatin may serve as an activity in the kidney remain to be investigated. On the basis of endogenous antagonist or inhibitor of TGF-␤ and VEGF in the previous observations that reagents or proteins that block kidney, and decreased angiostatin levels in diabetes may con- VEGF and TGF-␤ are beneficial for DN treatment, our study J Am Soc Nephrol 17: 475–486, 2006 Angiostatin in Diabetic Nephropathy 485 suggests that angiostatin should have therapeutic potential in suppressed by an orally effective beta-isoform-selective DN. inhibitor. Diabetes 46: 1473–1480, 1997 14. 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See related editorial, “Antiangiogenic Therapy in Diabetic Nephropathy,” on pages 325–327.