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Published ahead of print on January 4, 2006
J Am Soc Nephrol 17: 475-486, 2006
© 2006 American Society of Nephrology
doi: 10.1681/ASN.2005020217
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Pathophysiology of Renal Disease and Progression

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

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; jian-xing-ma{at}ouhsc.edu

Received for publication February 25, 2005. Accepted for publication November 25, 2005.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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-beta1, 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-beta1 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-beta1–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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Diabetic nephropathy (DN) is one of the most devastating microvascular complications of diabetes as well as the leading cause of ESRD in the United States (1,2). Approximately 20 to 40% of the patients with type 1 diabetes and 10 to 20% of those with type 2 diabetes develop nephropathy (3). The earliest pathologic changes of DN are characterized by glomerular hypertrophy, the thickening of glomerular basement membrane, and expanded extracellular matrix (ECM), which lead to glomerular hyperfiltration and microalbuminuria (2,4). Although intensified controls of hyperglycemia, BP, and hyperlipidemia reduce the risks of DN, they do not sufficiently prevent the progression from microalbuminuria to overt nephropathy in patients with diabetes (1,5,6).

Growth factors play an important role in the pathogenesis of DN (7). TGF-beta has been recognized as a major modulator of DN (8). Overexpression of TGF-beta in diabetic glomeruli is believed to contribute to the ECM accumulation by increasing synthesis and decreasing degradation of ECM proteins such as fibronectin and collagen (9–12). Another important growth factor involved in DN is vascular endothelial growth factor (VEGF). VEGF is a major angiogenic factor and a vascular permeability factor (13,14). VEGF expression has been shown to increase at the early stage of DN in both patients with diabetes and diabetic animal models (7,15). Blockade of VEGF bioactivity for 6 wk abolished glomerular hyperfiltration in streptozotocin (STZ)-induced diabetic rats (16).

The exact mechanisms underlying how these pathologic factors induce nephropathy in diabetes are largely unexplored. Accumulating evidence suggests that inflammation plays a crucial role in DN (17–20). Upregulated expression of proinflammatory cytokines, such as TNF-{alpha}, monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), and IL-18, is closely associated with renal functional damage (21–24). Knockout of ICAM-1 abolished diabetes-induced increase in urinary albumin excretion (UAE), glomerular hypertrophy, and mesangial matrix expansion, suggesting that inflammation may be partially responsible for the renal injury in diabetes (19).

Angiostatin, a proteolytic fragment (kringle 1 to 4) of plasminogen, was first identified in the serum and urine of tumor-bearing animals (25). Angiostatin is a potent angiogenic inhibitor that specifically inhibits proliferation and induces apoptosis in vascular endothelial cells (26). In vivo studies have shown that angiostatin efficiently arrests tumor growth and metastasis and also suppresses hypoxia-induced retinal neovascularization (27,28). Recently, we showed that angiostatin reduces retinal vascular leakage in the STZ-induced diabetic rat model and oxygen-induced retinopathy model (29). Therapeutic laser photocoagulation increases angiostatin levels in the vitreous of patients with diabetic retinopathy (DR) (30). These data suggest that decreased expression of angiostatin might be associated with the pathogenesis of DR. However, the implication of angiostatin in DN has not been established. In this study, we investigated the function of angiostatin in regulation of TGF-beta and VEGF in renal cells and measured angiostatin levels in diabetic rat kidney.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Brown Norway (BN) rats were purchased from Charles River Laboratories (Wilmington, MA). Care, use, and treatment of all animals in this study were in strict agreement with the guidelines set forth by the University of Oklahoma.

Induction of Experimental Diabetes
Diabetes was induced by an intraperitoneal injection of STZ (Sigma, St. Louis, MO) at 50 mg/kg body wt into 8-wk-old BN rats after an overnight fasting. Blood glucose levels were measured at 48 h after STZ injection. The animals with blood glucose >350 mg/dl were used as diabetic rats.

Cell Culture
Primary human glomerular mesangial cell (HMC) culture was described previously (31). Cells of passages 6 to 10 were used in the experiments. An immortalized mouse podocyte cell line was a gift from Dr. Peter Mundel (Albert Einstein College of Medicine, Bronx, NY) and maintained as documented previously (32). After reaching 80% confluence, cells were exposed to 0.5% FBS for 12 h followed by the treatment with desired reagents.

Western Blot Analysis of Angiostatin, TGF-beta1, VEGF, and ICAM-1
The kidney tissue was homogenized and centrifuged at 4°C. The protein concentration in the supernatant was measured with the BioRad DC protein assay (BioRad Laboratories, Hercules, CA). Fifty micrograms of protein from each sample was blotted by an anti-angiostatin or anti–TGF-beta1 antibody (R&D Systems, Minneapolis, MN). The same membranes were stripped and reblotted by anti-VEGF and anti–ICAM-1 (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies.

Determination of Matrix Metalloproteinase-2 mRNA Levels by Real-Time Reverse Transcription–PCR
Primers specific for matrix metalloproteinase-2 (MMP-2; forward 5'-ggccaactacaacttcttcc-3', reverse 5'-ccatcatggattcgagaaaa-3') were used for real-time reverse transcription–PCR (RT-PCR). The PCR was performed using GeneAmp RNA PCR kit and SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). The efficiency of real-time 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 calculated as follows: mRNA levels (percent of control) = 2{Delta}({Delta}CT) with {Delta}CT = CT, MMP-2 – CT, 18S RNA and {Delta}({Delta}CT) = {Delta}CT, normal sample – {Delta}CT, STZ-diabetic sample.

MMP-2 Activity Assay by Gelatin Zymography
Gelatinolytic activity of MMP-2 was analyzed by gelatin zymography (BioRad Laboratories) following the manufacturer’s protocol. Fifteen micrograms of tissue extracts or 20 µL of culture medium was applied to a precast 10% polyacrylamide gel with 1 mg/ml gelatin. After renaturation for 1 h and development overnight, the gel was stained with SimplyBlue SafeStain (BioRad) and photographed with Imager (Syngene, Cambridge, UK).

Intravenous Delivery of Adenovirus-Expressing Angiostatin
The adenovirus-expressing human angiostatin (Ad-Ang) contains human plasminogen kringle 1 to 4 under the control of the cytomegalovirus (CMV) promoter. The adenovirus expressing green fluorescence protein (Ad-GFP) contains a GFP gene under the control of the CMV promoter. Both of the viral vectors were purchased from Qbiogene (Montreal, QC, Canada). Diabetic rats were randomly assigned to three groups 1 wk after the STZ injection. Group 1 received no injection (n = 5), and groups 2 and 3 received an intravenous injection of Ad-Ang (n = 7) and Ad-GFP as controls (n = 5), respectively, at a dose of 4 x 1010 viral particles per rat.

Evaluation of Rat Microalbuminuria
The 24-h urine collected from each rat was centrifuged at 2000 x g for 5 min. The urine creatinine levels were determined using the QuantiChrom Creatinine Assay Kit (BioAssay Systems, Hayward, CA), following the manufacturer’s protocol. The concentration of urine albumin was measured by ELISA (Bethyl Laboratories Inc., Montgomery, TX). The UAE was normalized by creatinine excretion and expressed as mg albumin/mg creatinine in 24-h urine.

Quantification of Glomerular Volume and Glomerular Cell Numbers
The kidneys were fixed in 4% formaldehyde solution and paraffin-embedded, and 4-µm sections were cut. The sections were stained with Masson’s trichrome staining and read by two observers who were unaware of experimental protocol under a microscope (33). The glomerular areas were measured using SPOT Advanced Software (Diagnostic Instruments, Inc., Sterling Heights, MI) and averaged from 120 to 150 glomeruli per kidney (34). The glomerular volume was calculated by the formula VG = area1.5x 1.38/1.1 (35). The cell numbers in glomeruli were counted, and the average glomerular cell number was obtained from 100 glomeruli per kidney.

Measurement of VEGF, TGF-beta1, Pigment Epithelium–Derived Factor, MCP-1, and Fibronectin Protein Level by ELISA
The protein levels of VEGF, TGF-beta1, pigment epithelium–derived factor (PEDF), and fibronectin in the cell culture medium or in the kidney tissue homogenate were quantified using the commercial Quantikine VEGF or TGF-beta1 ELISA Kit (R&D Systems, Minneapolis, MN), PEDF ELISA kit (Chemicon Inc., Temecula, CA), and fibronectin ELISA (Assaypro, Winfield, MO), respectively, according to the manufacturer’s protocols. The amount of MCP-1 was measured by a MCP-1 ELISA kit (Chemicon Inc.).

Smad Nuclear Translocation Assay
Primary HMC were cultured on four-chamber slides (Nalge Nunc International Corp., Naperville, IL) to reach 80% confluence. After exposure to 2.5 ng/ml TGF-beta1 with or without 100 nM angiostatin for 1 h, the cells were fixed immediately with 4% paraformaldehyde. The cells were incubated with anti–Smad2/3 antibody (1:200; Upstate USA, Inc., Lake Placid, NY) for 2 h and then incubated with cy3-conjugated donkey anti-rabbit antibody for 1 h. The slide was visualized under a fluorescence microscope (Olympus, Hamburg, Germany).

Statistical Analyses
Statistical analysis used t test. Statistical difference was considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Angiostatin Is Generated in Normal Rat Kidney and Decreased in Diabetic Kidney
Western blot analysis showed that high levels of plasminogen and its proteolytic fragments exist in the kidney and liver (Figure 1A). Two forms of angiostatin with apparent molecular weights of approximately 50 and 38 kD were identified in the kidney, but only the 38-kD form was found in the liver (Figure 1A). In the retina, there were only low levels of plasminogen but not its proteolytic fragments (Figure 1A).


Figure 1
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  		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 µg) 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-beta1 in the same kidneys as those used in B. The membrane was stripped and reblotted with an anti–beta-actin antibody.

 
Angiostatin levels were determined in the kidney from BN rats with diabetes for 6 wk, which had developed polyuria and microalbuminuria (data not shown). The results showed that angiostatin levels were drastically decreased in diabetic kidney (Figure 1, B and C). In contrast, the same gel showed that the intact plasminogen levels were significantly higher in diabetic kidney than that in normal control, demonstrating a decreased proteolysis of plasminogen leading to a reduced release of angiostatin (Figure 1, B and C). In the same samples, however, TGF-beta1 levels were significantly increased in the diabetic kidneys (Figure 1D).

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


Figure 2
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  		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 µg) 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 ± SD, n = 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-beta1 (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.

 
To explore further the possible mechanism that is responsible for the decrease of angiostatin in diabetic kidney, we investigated the expression of MMP-2, which was recognized as a major protease responsible for the release of angiostatin from plasminogen (38). Real-time RT-PCR demonstrated that mRNA levels of MMP-2 were drastically decreased in both the cortex and the medulla from rats with 6 wk of diabetes (Figure 2B). The gelatin zymography showed that enzymatic activities of MMP-2 were also significantly lower in diabetic cortex and medulla (Figure 2C). These results suggest that the decreased expression of MMP-2 might be responsible, at least in part, for the decreased angiostatin levels in diabetic kidney.

To identify the cause of the MMP-2 decrease in diabetic kidney, we examined the effects of high glucose and TGF-beta1 on MMP-2 expression in cultured glomerular cells. As endothelial cells in the glomeruli express only very low levels of MMP, we first compared the MMP-2 activity in the cultured HMC and the podocyte cell line (39). The results showed that the MMP-2 activity in the conditioned medium from HMC was 10-fold higher than that from podocytes after a 48-h culture (P < 0.01; Figure 2D), consistent with the previous study showing the high expression of MMP-2 in mesangial cells but not in podocyte or endothelial cells (40). In HMC that were treated with 30 mM glucose for 72 h, MMP-2 activities in the conditioned medium were significantly lower than those in the normal control (5 mM glucose) or mannitol osmotic control (5 mM glucose + 25 mM mannitol; Figure 2E). TGF-beta1 (5 ng/ml) treatment for 48 h also significantly decreased the MMP-2 secretion from HMC (Figure 2E). The decrease of MMP-2 activity that was induced by high glucose was also observed in podocytes (Figure 2F). These results suggest that both hyperglycemia and overexpression of TGF-beta contribute to the decrease of angiostatin in diabetic kidney.

Decrease of Albuminuria and Renal VEGF and TGF-beta Levels and Attenuation of Glomerular Hypertrophy in Diabetic Rats Treated with Ad-Ang
Ad-Ang and the same amount of the control virus, Ad-GFP, were injected separately into the diabetic rats 1 wk after the onset of diabetes. The UAE was evaluated at 2 wk after the adenovirus delivery. The results showed that in 3-wk diabetic rats without virus injection, the UAE was increased by 30-fold over that in age-matched nondiabetic controls (P < 0.01, n = 5; Figure 3A). No significant difference in the UAE was observed between the diabetic rats that received Ad-GFP injection or not, indicating that the injection of adenovirus vector did not affect albuminuria. The UAE in the rats that received Ad-Ang injection was significantly lower than that in the rats that received control virus injection and in untreated diabetic rats (P < 0.01, n = 5; Figure 3A), suggesting that angiostatin protects the kidney from early diabetic injury.


Figure 3
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  		Figure 3. Effect of angiostatin on microalbuminuria, glomerular hypertrophy, and overproduction of renal VEGF and TGF-beta 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 ± SD, n = 5). (B) The glomerular volume was measured in the kidneys at 3 wk after the Ad-Ang delivery (mean ± SD, n = 5). An average of 120 to 150 glomeruli were measured in each kidney. The TGF-beta1 (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-beta1 levels in the rats that received Ad-Ang injection were significantly lower than that in the rats that received Ad-GFP injection (P < 0.05, n = 3).

 
Glomerular hypertrophy has been well characterized in the early STZ-induced diabetes and is closely linked to glomerular hyperfiltration and microalbuminuria in rats (34,41,42). We evaluated the effect of angiostatin on glomerular volume in diabetic kidneys. The results showed that at 4 wk after the onset of diabetes, the glomerular volume was increased by 30% in diabetic rats when compared with age-matched nondiabetic controls (P < 0.01, n = 5; Figure 3B). Angiostatin treatment successfully attenuated the glomerular hypertrophy (P < 0.01, n = 5; Figure 3B). We also counted the glomerular cell numbers in experimental and control groups. The glomerular cell numbers, however, did not show any significant difference between the diabetic kidneys and normal control (data not shown). There was also no difference in cell numbers between the Ad-Ang–treated group and control virus–treated groups (data not shown).

As TGF-beta is a major pathogenic factor responsible for the glomerular hypertrophy in the kidney in diabetic rat models (9,12,32), we next examined the effect of Ad-Ang on TGF-beta1 expression in diabetic kidneys. The results showed that the renal TGF-beta1 levels were significantly increased in the diabetic kidney with or without the Ad-GFP virus treatment. Ad-Ang delivery significantly decreased TGF-beta levels in diabetic kidneys (P < 0.05, n = 5; Figure 3C).

In addition, we determined the effect of Ad-Ang on VEGF levels in diabetic rat kidneys, as VEGF is another known pathogenic factor in DN. The results showed that at 3 wk after the onset of diabetes, the renal VEGF levels were significantly elevated, consistent with the increase of TGF-beta1 levels and glomerular hypertrophy. The Ad-Ang treatment significantly reduced the VEGF levels in the kidney of diabetic rats (P < 0.05, n = 5; Figure 3D).

Angiostatin Inhibits High-Glucose–Induced Overexpression of VEGF and TGF-beta in HMC
We determined the direct effect of angiostatin on VEGF expression in cultured glomerular cells. After incubation of HMC with a high-glucose (30 mM) medium for 48 h, secreted VEGF levels were significantly increased over the mannitol control (Figure 4A). Angiostatin decreased high-glucose–induced VEGF secretion to the level of the mannitol control. This angiostatin effect was also observed at 72 and 96 h of the treatment (Figure 4A).


Figure 4
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  		Figure 4. Angiostatin-induced downregulation of VEGF and TGF-beta expression in HMC that were cultured in high glucose. HMC were incubated with high glucose (30 mM) in the absence or presence of 100 nM angiostatin; 5 mM glucose + 25 mM mannitol was used as the osmotic control. The medium was harvested at 48, 72, and 96 h after the incubation. The VEGF level (A) and TGF-beta1 level (B) in the medium were measured by ELISA. The results were normalized by total protein concentration in the medium and expressed as pg/mg total protein (mean ± SD, n = 3). Values that are statistically different from the normal controls are indicated by *P < 0.05 or **P < 0.01. Values that are statistically different from the high glucose alone are indicated by {dagger}P < 0.05 or {ddagger}P < 0.01.

 
The direct effect of angiostatin on TGF-beta1 secretion from HMC was also evaluated in cultured HMC. After 48 h of incubation with high glucose in the absence or presence of angiostatin, TGF-beta1 levels in the culture medium were comparable to that in the cells that were exposed to mannitol for osmotic controls (Figure 4B). After 72- and 96-h incubations, the TGF-beta1 secretion was significantly increased by high glucose over those in the mannitol control (Figure 4B). Angiostatin significantly inhibited high-glucose–induced TGF-beta1 secretion at 72 and 96 h of the treatment (Figure 4B).

Angiostatin Decreases High-Glucose–and TGF-beta1–Induced MCP-1 Secretion in HMC
MCP-1 is one of the most important proinflammatory chemokines implicated in the pathogenesis of DN (17,43,44). High glucose is known to upregulate MCP-1 expression via the NF-{kappa}B activation in cultured renal mesangial cells (45). In our study, we examined the effects of angiostatin on MCP-1 secretion that was induced by high glucose and TGF-beta1. After incubation with high glucose for 48 h, the MCP-1 secretion was increased by 2.4-fold, when compared with that of the mannitol control (Figure 5A). Angiostatin from 2 to 250 nM decreased MCP-1 secretion in a concentration-dependent manner.


Figure 5
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  		Figure 5. Inhibitory effect of angiostatin on monocyte chemoattractant protein-1 (MCP-1) secretion in HMC. HMC were incubated with 5 ng/ml TGF-beta1 (A) or 30 mM glucose (B) in the absence or presence of different concentrations of angiostatin (0.4 to 250 nM) for 48 h. MCP-1 levels in the medium were measured by ELISA. The results were normalized by total protein concentrations in the medium and expressed as pg/mg of total protein (mean ± SD, n = 3). Values that are statistically different from the normal glucose controls are indicated by **P < 0.01. Values that are statistically different from the cells that were treated with high glucose or TGF-beta1 are indicated by {dagger}P < 0.05 or {ddagger}P < 0.01

 
Similarly, TGF-beta1 (5 ng/ml) treatment for 48 h significantly increased the MCP-1 secretion in HMC (Figure 5B). Angiostatin (2 to 50 nM) blocked TGF-beta1–induced MCP-1 increase in a concentration-dependent manner, suggesting an anti-inflammatory effect of angiostatin.

Angiostatin Prevents High-Glucose–Induced Downregulation of PEDF in Cultured HMC
PEDF is an endogenous angiogenic inhibitor that has been implicated in DR (46–48). Recently, we reported that PEDF acts as an endogenous inhibitor of TGF-beta and VEGF in the kidney (31). In our study, we determined the effect of angiostatin on PEDF secretion in cultured HMC that were insulted by TGF-beta1 and angiotensin II, two common pathologic factors of DN (9,49).

After incubation with 5 ng/ml TGF-beta1 for 48 h, PEDF secretion was decreased by three-fold compared with that in the control cells (P < 0.001, n = 4; Figure 6A). Angiostatin efficiently inhibited the TGF-beta1–induced PEDF decrease in a concentration-dependent manner (Figure 6A). Moreover, under normal conditions, 50 nM angiostatin significantly increased PEDF secretion, suggesting that angiostatin may be a potential positive regulator of PEDF secretion (Figure 6A).


Figure 6
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  		Figure 6. Angiostatin-induced upregulation of pigment epithelium–derived factor (PEDF) expression in HMC. Cultured HMC were challenged with 5 ng/ml TGF-beta1 (A) or 50 ng/ml angiotensin II (B) in the absence or presence of different doses of angiostatin (0.4 to 250 nM) for 48 h. PEDF levels in the medium were measured by ELISA. The results were normalized by total protein concentrations in the medium and expressed as ng/mg total protein (mean ± SD, n = 3). Values that are statistically different from the normal controls are indicated by *P < 0.05 or **P < 0.01. Values that are statistically different from TGF-beta–or angiotensin II–treated are indicated by {dagger}P < 0.05 or {ddagger}P < 0.01.

 
Angiotensin II has been shown as a crucial factor inducing glomerulosclerosis in DN through stimulating ECM production and inhibiting ECM degradation (49). Our study showed that 50 ng/ml angiotensin II decreased PEDF secretion in HMC. Angiostatin (2 to 250 nM) effectively prevented the PEDF decrease induced by angiotensin II (Figure 6B).

Angiostatin Blocks High-Glucose–and Angiotensin II–Induced Fibronectin Secretion from HMC
In DN, the overproduction of ECM proteins, such as fibronectin and collagen, is a major causative factor that is responsible for the glomerular hyperfiltration and mesangial expansion in diabetic kidneys (50). In cultured primary HMC, an exposure to high glucose (30 mM) for 48 h led to significant increases of fibronectin secretion (Figure 7A). At concentrations from 10 to 250 nM, angiostatin decreased the fibronectin secretion in a concentration-dependent manner (Figure 7A). In the cells that were exposed to 50 ng/ml angiotensin II for 48 h, the fibronectin production was dramatically increased (Figure 7B). Angiostatin (10 to 250 nM) showed a concentration-dependent inhibition of fibronectin secretion induced by angiotensin II (Figure 7B).


Figure 7
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  		Figure 7. Angiostatin blocks high-glucose–induced fibronectin secretion from HMC. HMC were incubated with 30 mM glucose (A) or 50 ng/ml of angiotensin II (B) 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. The fibronectin level in the serum (0.5%) was measured and subtracted. The results were normalized by total protein concentrations in the medium and expressed as µg/mg total protein (mean ± SD, n = 3). Values that are statistically different from the normal controls are indicated by **P < 0.01. Values that are statistically different from cells that were exposed to high glucose or angiotensin II are indicated by {ddagger}P < 0.01.

 
Angiostatin Suppresses TGF-beta1–Induced Fibronectin Production via Blocking Smad2/3 Activation in HMC
As a crucial mediator of ECM production and accumulation, TGF-beta strongly stimulated HMC to produce fibronectin. After incubation with TGF-beta1 for 48 h, the fibronectin secretion from HMC was increased by four-fold over the control. In the presence of different concentrations of angiostatin (0.4 to 250 nM), the effect of TGF-beta1 was abolished in an angiostatin concentration-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 antibody specific for cellular fibronectin the results of fibronectin changes that were obtained from ELISA (data not shown).


Figure 8
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  		Figure 8. Angiostatin blocks TGF-beta1 function via blockade of Smad2/3 activation in HMC. (A) HMC were treated with 5 ng/ml TGF-beta1 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 µg/mg of total protein (mean ± SD, n = 3). Values that are statistically different from the normal controls are indicated by **P < 0.01. Values that are statistically different from cells that were treated with TGF-beta1 without angiostatin are indicated by {dagger}P < 0.05 or {ddagger}P < 0.01. (B) HMC were incubated with 5 ng/ml TGF-beta 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-beta1 (B-b), when compared with that in the control cells without TGF-beta1 treatment (B-a). (B-c) Angiostatin (100 nM) effectively blocked the TGF-beta1–induced upregulation and translocation of Smad2/3. Magnification, x400 in B.

 
To explore further the possible mechanism underlying the effect of angiostatin on TGF-beta1–induced fibronectin production, we determined whether the inhibitory effect of angiostatin on TGF-beta is through blocking Smad activation, which is a major signaling pathway mediating TGF-beta functions. HMC were incubated with TGF-beta1 (5 ng/ml) in the absence or presence of 100 nM angiostatin for 1 h followed by immunocytochemistry assay with an anti-Smad2/3 antibody. The results showed that when compared with control cells (Figure 8B-a), TGF-beta1 stimulated Smad2/3 expression and translocation from the cytoplasm to the nuclei, a critical step in TGF-beta function (Figure 8B-b). Treatment with 100 nM angiostatin significantly blocked the nuclear translocation of Smad2/3 induced by TGF-beta1 (Figure 8B-c).

Angiostatin Does not Affect Growth of HMC
To determine whether the inhibitory effect of angiostatin on fibronectin production is through affecting the proliferation of mesangial cells, we examined the effect of angiostatin on HMC growth. The results showed that angiostatin did not affect the HMC viability under either high-glucose condition (30 mM) or normal-glucose condition (5 mM; Figure 9), suggesting that the angiostatin-induced decreases of fibronectin and TGF-beta1 levels are not a result of reduced cell numbers.


Figure 9
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  		Figure 9. Angiostatin had no effect on cell proliferation in HMC. The MTT assay was used to determine the viable HMC number after treatments with different concentrations of angiostatin for 3 d under both normal-glucose (A) and high-glucose (B) conditions. The results showed that angiostatin had no effect on viable cell numbers of HMC.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Angiostatin is known as an inhibitor of angiogenesis, tumor growth, and metastasis (25,28,51,52). This study identified a novel function of angiostatin: Inhibiting high-glucose–induced overexpression of VEGF and TGF-beta1 and also suppressing inflammation and fibrosis in kidney cells under diabetic stresses. Moreover, we demonstrate that generation of endogenous angiostatin is significantly decreased in the kidney of a diabetic rat model, and delivery of angiostatin significantly alleviates functional abnormalities in the kidney of diabetic rats. These findings suggest that angiostatin may serve as an endogenous inhibitor of inflammation and fibrosis in the normal kidney, and the decrease of the angiostatin generation in the diabetic kidney may contribute to pathologic changes of DN. This study for the first time reveals the implication of angiostatin in DN.

Angiostatin was first identified as internal fragments of plasminogen in the serum and urine of tumor-bearing animals (25). Although angiostatin was given a single name, in fact angiostatin refers to several fragments of plasminogen, such as kringle 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 homogenate from normal BN rats, we observed two forms of angiostatin at molecular weights of approximately 50 and 38 kD, consistent with the two forms of angiostatin reported by Basile et al. (53) recently. Only the 38-kD form was observed in the liver. No angiostatin or other proteolytic fragments of plasminogen were observed in the retina, likely because of the low abundance of plasminogen in the retina. These results suggest that the generation of angiostatin is tissue specific.

Previous studies have shown that angiostatin, as an endogenous angiogenic inhibitor, is implicated in DR (29). In this study, we demonstrated that angiostatin levels were decreased in the kidney of rats with 6 wk of diabetes, which are known to have functional and structural abnormalities of DN, including polyuria, microalbuminuria, and renal inflammation (16). This result for the first time suggests a potential role of decreased angiostatin levels in the development or progression of DN. Moreover, our results demonstrate that plasminogen levels, in contrast to the decreased angiostatin levels, are significantly higher in the diabetic kidney, suggesting that the proteolytic release of angiostatin from plasminogen, rather than the expression of the plasminogen gene, is deficient in diabetes. This conclusion is further supported by the observation that both the expression and the activity of MMP-2 are suppressed in diabetic kidney, as MMP-2 has been shown to release angiostatin from plasminogen (38).

Although endothelial cells in the retina are recognized as a major source of MMP, previous studies showed that MMP are expressed mainly in mesangial cells and podocytes in the glomeruli (39,40). In the three types of glomerular cells, MMP-2 mRNA levels in the podocytes and endothelial cells were only 33 and 18%, respectively, of that in the mesangial cells (40). Our study showed that the MMP-2 activity in conditioned medium from HMC culture was 10-fold higher than that from mouse podocytes, suggesting a predominant role of mesangial cells in MMP-2 production. Moreover, our studies showed that exposure of the cells to high glucose or TGF-beta increased the MMP-2 activity in both mesangial cells and podocytes, suggesting that the decrease of MMP-2 expression and subsequent decrease of angiostatin levels in the diabetic kidney could be induced by hyperglycemia and the increase of TGF-beta levels in DN.

To investigate the function of angiostatin in the kidney, we delivered recombinant angiostatin via an adenovirus-mediated gene. The angiostatin gene delivery indeed reduced UAE in diabetic rats almost to the normal level, whereas the control adenovirus that expressed GFP did not affect the microalbuminuria under the same conditions, suggesting a potent effect of angiostatin on the inhibition of microalbuminuria. As microalbuminuria has been shown to be closely linked with glomerular hypertrophy in the early stage of DN, we further determined the effect of angiostatin on glomerular hypertrophy (34,41,42,54,55). Consistent with the reduction of UAE, the glomerular hypertrophy was significantly attenuated in angiostatin-treated rats. Furthermore, we investigated the effect of angiostatin on the expression of VEGF and TGF-beta, which are recognized as the major pathogenic factors responsible for the glomerular hypertrophy and proteinuria (15,34). The results showed that the kidney VEGF and TGF-beta levels were significantly decreased in angiostatin-treated rats, suggesting that angiostatin is a potent inhibitor of VEGF and TGF-beta expression in the kidney.

The potent effect of angiostatin on the inhibition of VEGF and TGF-beta in diabetic kidney was confirmed further by in vitro studies. In cultured HMC, angiostatin efficiently blocked high-glucose–induced overexpression of TGF-beta and VEGF. Moreover, angiostatin inhibited the function of TGF-beta, i.e., blocking TGF-beta–induced MCP-1 and fibronectin expression. This effect is at least partially mediated by the inhibition of Smad activation, a major signaling pathway that mediates TGF-beta activities. These results further suggest that angiostatin may serve as an endogenous antagonist or inhibitor of TGF-beta and VEGF in the kidney, and decreased angiostatin levels in diabetes may contribute to the overexpression of these two pathogenic factors of DN.

PEDF is recognized as an anti-angiogenic factor and neurotrophic 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-beta 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-{alpha}, 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-beta–induced MCP-1 secretion in mesangial cells, suggesting that angiostatin inhibits inflammation in DN. These results were consistent with the recent report about the anti-inflammatory effect of angiostatin (56).

Overproduction of ECM proteins and mesangial matrix expansion are the early characteristics of DN that contribute to microalbuminuria (1,57). As mesangial cells are the major producer 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 glucose concentration, angiotensin II, and TGF-beta. The results showed that angiostatin blocked the fibronectin overproduction that is induced by all of these stressors. At the same concentration, however, angiostatin had no effect on mesangial cell growth, suggesting that the inhibition of fibronectin production is not a result of changed viable cell numbers. This result is consistent with our in vivo studies showing that the adenovirus-delivered angiostatin treatment ameliorated hypertrophy of glomeruli but not the hyperplasia in the diabetic kidneys.

Most previous studies of angiostatin focused primarily on its therapeutic potential in tumor and retinal neovascularization (51,52,58,59). The function of angiostatin in the kidney has not been studied previously. Our results showed that angiostatin blocks the expression and function of VEGF and TGF-beta but enhances the expression of endogenous protective factor PEDF. It also inhibits inflammation and ECM production under diabetic stresses. Therefore, the decreased generation of angiostatin in diabetic kidney may contribute to the pathologic changes of DN. Although our study suggests that ATP synthase is a possible receptor for angiostatin in mesangial cells (data not shown), the mechanisms that are responsible for the angiostatin activity in the kidney remain to be investigated. On the basis of previous observations that reagents or proteins that block VEGF and TGF-beta are beneficial for DN treatment, our study suggests that angiostatin should have therapeutic potential in DN.


   Acknowledgments
 
This study was supported by National Institutes of Health grants EY015650 and EY012231, a grant from the Oklahoma Center for the Advancement of Science and Technology, Juvenile Diabetes Research Foundation grant 1-2003-627, and American Diabetes Association grant 1-05-RA-75, and the Center of Biomedical Research Excellence to vision program at the University of Oklahoma Health Sciences Center.

We thank the Imaging Core Facility at the Oklahoma Medical Research Foundation for the technical assistance in histologic analysis.


   Footnotes
 
Published online ahead of print. Publication date available at www.jasn.org.

S.X.Z. and J.J.W. contributed equally to this study.


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