Inhibition Of Connective Tissue Growth Factor Research Articles

Combination of Tripterygium wilfordii Hook F and angiotensin receptor blocker synergistically reduces excretion of urinary podocytes in patients with type 2 diabetic kidney disease

The aim of this study was to investigate whether Tripterygium wilfordii Hook F (TwHF) and irbesartan could synergistically affect the urinary excretion of podocytes and proteins in type 2 diabetic kidney disease (DKD) patients and the underlying mechanisms. Forty DKD patients were divided into a DI group (DKD patients treated with irbesartan alone) and a DTI group (DKD patients treated with Tripterygium wilfordii Hook F and irbesartan). Urinary podocytes were observed by immunofluorescence. Urinary levels of connective tissue growth factor (CTGF) and transforming growth factor-β1 (TGF-β1) were detected by enzyme-linked immunosorbent assay. Immunofluorescence indicated that shed podocytes were not detected in urine samples of normal controls, whereas the detection rate of urinary podocytes was 82.5% in DKD patients. Urinary CTGF and TGF-β1 levels were significantly higher in urinary podocyte-positive DKD patients than in urinary podocyte-negative patients. Furthermore, urinary podocyte excretion was closely correlated with urinary protein excretion and urinary CTGF/TGF-β1 levels. Treatments with TwHF and irbesartan significantly reduced the urinary excretion of proteins and podocytes, and decreased the urinary levels of CTGF and TGF-β1. Our results suggest that urinary podocyte excretion might serve as a predictor for DKD progression. TwHF/irbesartan combination could reduce the urinary excretion of proteins and podocytes synergistically in DKD patients, which might result from the synergistic inhibition of CTGF and TGF-β1 in urine.

Astragaloside IV inhibits renal tubulointerstitial fibrosis by blocking TGF-β/Smad signaling pathway in vivo and in vitro.

Astragaloside IV (AS-IV) is a major active ingredient from Radix astragali, which has been considered as a renoprotective agent; however, its molecular mechanisms are unclear. Thus, we designed to investigate the renoprotective effects and mechanisms of AS-IV in rat model of renal fibrosis induced by unilateral ureteral obstruction (UUO) in vivo and TGF-β1-stimulated rat renal fibroblasts (NRK-49F) in vitro. Sprague-Dawley rats were randomly divided into six groups: sham operation, UUO, UUO/AS-IV (3.3, 10, 33 mg·kg(-1)·d(-1)), and UUO/enalapril (4 mg·kg(-1)·d(-1)). Renal function, tubulointerstitial damage index score, extracellular matrix (ECM) deposition, and the expressions of TGF-β1, connective tissue growth factor (CTGF), α-SMA, fibronectin, collagen I, III, Smad2/3, phosphorylated-Smad2/3, and Smad7 were measured. In addition, the expressions of CTGF, α-SMA, fibronectin, collagen I, III, Smad2/3, phosphorylated-Smad2/3, and Smad7 were measured in TGF-β1-stiumlated NRK-49F cell line. AS-IV significantly decreased UUO-induced renal fibrosis and functional impairment, which are associated with inhibition of TGF-β1, CTGF, α-SMA, and collagen matrix expression, and a decrease in serum creatinine and urea nitrogen. The renoprotective effects of AS-IV on fibrosis were associated with up-regulation of Smad7, thereby blocking up-regulations of TGF-β1, CTGF, and α-SMA, and activation of phosphorylated-Smad2/3. These effects were further conformed in NRK-49F cell line stimulated by TGF-β1. Moreover, knockdown of Smad7 gene in NRK-49F cells was able to prevent AS-IV-induced inhibition to Smad2/3 signaling activation, expression of CTGF, α-SMA, and ECM proteins in response to TGF-β1. Renal tubulointerstitial fibrosis was attenuated by treatment with AS-IV, which was closely related to induction of Smad7, thereby inhibiting TGF-β/Smad signaling.

Connective Tissue Growth Factor Inhibition Attenuates Left Ventricular Remodeling and Dysfunction in Pressure Overload–Induced Heart Failure

Connective tissue growth factor (CTGF) is involved in the pathogenesis of various fibrotic disorders. However, its role in the heart is not clear. To investigate the role of CTGF in regulating the development of cardiac fibrosis and heart failure, we subjected mice to thoracic aortic constriction (TAC) or angiotensin II infusion, and antagonized the function of CTGF with CTGF monoclonal antibody (mAb). After 8 weeks of TAC, mice treated with CTGF mAb had significantly better preserved left ventricular (LV) systolic function and reduced LV dilatation compared with mice treated with control immunoglobulin G. CTGF mAb-treated mice exhibited significantly smaller cardiomyocyte cross-sectional area and reduced expression of hypertrophic marker genes. CTGF mAb treatment reduced the TAC-induced production of collagen 1 but did not significantly attenuate TAC-induced accumulation of interstitial fibrosis. Analysis of genes regulating extracellular matrix proteolysis showed decreased expression of plasminogen activator inhibitor-1 and matrix metalloproteinase-2 in mice treated with CTGF mAb. In contrast to TAC, antagonizing the function of CTGF had no effect on LV dysfunction or LV hypertrophy in mice subjected to 4-week angiotensin II infusion. Further analysis showed that angiotensin II-induced expression of hypertrophic marker genes or collagens was not affected by treatment with CTGF mAb. In conclusion, CTGF mAb protects from adverse LV remodeling and LV dysfunction in hearts subjected to pressure overload by TAC. Antagonizing the function of CTGF may offer protection from cardiac end-organ damage in patients with hypertension.

Connective Tissue Growth Factor Is Involved in Structural Retinal Vascular Changes in Long-Term Experimental Diabetes

Early retinal vascular changes in the development of diabetic retinopathy (DR) include capillary basal lamina (BL) thickening, pericyte loss and the development of acellular capillaries. Expression of the CCN (connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed) family member CCN2 or connective tissue growth factor (CTGF), a potent inducer of the expression of BL components, is upregulated early in diabetes. Diabetic mice lacking one functional CTGF allele (CTGF+/−) do not show this BL thickening. As early events in DR may be interrelated, we hypothesized that CTGF plays a role in the pathological changes of retinal capillaries other than BL thickening. We studied the effects of long-term (6-8 months) streptozotocin-induced diabetes on retinal capillary BL thickness, numbers of pericytes and the development of acellular capillaries in wild type and CTGF+/− mice. Our results show that an absence of BL thickening of retinal capillaries in long-term diabetic CTGF+/− mice is associated with reduced pericyte dropout and reduced formation of acellular capillaries. We conclude that CTGF is involved in structural retinal vascular changes in diabetic rodents. Inhibition of CTGF in the eye may therefore be protective against the development of DR.

Inhibition of Connective Tissue Growth Factor Ameliorates Disease in a Murine Model of Rheumatoid Arthritis

We have shown that connective tissue growth factor (CTGF) plays an important role in the pathogenesis of rheumatoid arthritis (RA). This study was undertaken to evaluate the effects of blockade of the CTGF pathway on the development of collagen-induced arthritis (CIA) in mice. Arthritis was induced in DBA/1J mice by immunization with a combination of type II collagen (CII) and Freund's complete adjuvant. We evaluated the development of arthritis in mice with CIA left untreated versus treated with neutralizing anti-CTGF monoclonal antibody (mAb). Inhibition of CTGF in mice treated with neutralizing anti-CTGF mAb significantly ameliorated arthritis compared to the untreated mice with CIA. Serum levels of matrix metalloproteinase 3 were reduced by anti-CTGF mAb treatment. Moreover, blockade of CTGF decreased interleukin-17 expression on purified CD4+ T lymphocytes. Although the expression of the retinoic acid receptor-related orphan receptor γt gene was not suppressed by anti-CTGF mAb treatment, that of interferon regulatory factor 4 (IRF-4) and IκBζ (Nfkbiz), which are other important molecules for the differentiation of Th17 cells, was suppressed. In addition, blockade of CTGF inhibited pathologic proliferation of T lymphocytes in response to CII restimulation in vitro. Moreover, aberrant osteoclastogenesis in mice with CIA was restored by anti-CTGF mAb treatment. Our findings indicate that blockade of CTGF prevents the progression of arthritis in mice with CIA. Anti-CTGF mAb treatment suppresses pathologic T cell function and restores aberrant osteoclastogenesis in mice with CIA. CTGF may become a new target for the treatment of RA.

Abstract 3379: Overexpression of CTGF is associated with chemoresistance in esophageal adenocarcinoma.

Abstract Esophageal adenocarcinoma is the most rapidly increasing solid malignancy with an overall 5-year survival of only 19%. Neoadjuvant chemoradiation is standard practice in most large centers but results in a complete pathologic response in only 21%, although this subset has an improved 5-year survival. In this study, connective tissue growth factor (CTGF), which is associated with increased cell migration, tissue invasion, and angiogenesis, was evaluated for its role in chemoresistance in esophageal adenocarcinoma. CTGF was found to be overexpressed in 33.3% (5 of 15; mean 3.1-fold) of esophageal adenocarcinomas on oligonucleotide microarray relative to Barrett's metaplasia. Elevated expression of CTGF was also found in 5/5 advanced stage IV esophageal adenocarcinomas on Western blot. Overexpression of CTGF was observed in 70.6% of chemoresistant esophageal adenocarcinomas (n=17) as determined using qRT-PCR and was significantly higher compared to chemo-naïve esophageal adenocarcinomas (n=102; p<0.0005). Stable inhibition with an shRNA against CTGF in the esophageal adenocarcinoma cell line Flo, which expresses endogenous CTGF, led to a significantly increased response to the chemotherapeutic agents 5-FU (9.4-fold increase), cisplatin (5.5-fold), and etoposide (4.4-fold) at 24 hours (p<0.005). These findings suggest that CTGF may be important in mediating chemoresistance in esophageal adenocarcinoma and that inhibition of CTGF could be useful in modulating chemoresistance to current neoadjuvant regimens in esophageal adenocarcinomas overexpressing CTGF. Citation Format: Zhuwen Wang, Lin Lin, Dafydd G. Thomas, Mark B. Orringer, Andrew C. Chang, David G. Beer, Jules Lin. Overexpression of CTGF is associated with chemoresistance in esophageal adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3379. doi:10.1158/1538-7445.AM2013-3379

CTGF is a therapeutic target for metastatic melanoma

Metastatic melanoma remains a devastating disease with a 5-year survival rate of less than five percent. Despite recent advances in targeted therapies for melanoma, only a small percentage of melanoma patients experience durable remissions. Therefore, it is critical to identify new therapies for the treatment of advanced melanoma. Here, we define connective tissue growth factor (CTGF) as a therapeutic target for metastatic melanoma. Clinically, CTGF expression correlates with tumor progression and is strongly induced by hypoxia through HIF-1 and HIF-2-dependent mechanisms. Genetic inhibition of CTGF in human melanoma cells is sufficient to significantly reduce orthotopic tumor growth, as well as metastatic tumor growth in the lung of severe combined immunodeficient (SCID) mice. Mechanistically, inhibition of CTGF decreased invasion and migration associated with reduced matrix metalloproteinase-9 expression. Most importantly, the anti-CTGF antibody, FG-3019, had a profound inhibitory effect on the progression of established metastatic melanoma. These results offer the first preclinical validation of anti-CTGF therapy for the treatment of advanced melanoma and underscore the importance of tumor hypoxia in melanoma progression.

Combination of roflumilast with a beta-2 adrenergic receptor agonist inhibits proinflammatory and profibrotic mediator release from human lung fibroblasts

BackgroundSmall airway narrowing is an important pathology which impacts lung function in chronic obstructive pulmonary disease (COPD). The accumulation of fibroblasts and myofibroblasts contribute to inflammation, remodeling and fibrosis by production and release of mediators such as cytokines, profibrotic factors and extracellular matrix proteins. This study investigated the effects of the phosphodiesterase 4 inhibitor roflumilast, combined with the long acting β2 adrenergic agonist indacaterol, both approved therapeutics for COPD, on fibroblast functions that contribute to inflammation and airway fibrosis.MethodsThe effects of roflumilast and indacaterol treatment were characterized on transforming growth factor β1 (TGFβ1)-treated normal human lung fibroblasts (NHLF). NHLF were evaluated for expression of the profibrotic mediators endothelin-1 (ET-1) and connective tissue growth factor (CTGF), expression of the myofibroblast marker alpha smooth muscle actin, and fibronectin (FN) secretion. Tumor necrosis factor-α (TNF-α) was used to induce secretion of chemokine C-X-C motif ligand 10 (CXCL10), chemokine C-C motif ligand 5 (CCL5) and granulocyte macrophage colony-stimulating factor (GM-CSF) from NHLF and drug inhibition was assessed.ResultsEvaluation of roflumilast (1-10 μM) showed no significant inhibition alone on TGFβ1-induced ET-1 and CTGF mRNA transcripts, ET-1 and FN protein production, alpha smooth muscle expression, or TNF-α-induced secretion of CXCL10, CCL5 and GM-CSF. A concentration-dependent inhibition of ET-1 and CTGF was shown with indacaterol treatment, and a submaximal concentration was chosen for combination studies. When indacaterol (0.1 nM) was added to roflumilast, significant inhibition was seen on all inflammatory and fibrotic mediators evaluated, which was superior to the inhibition seen with either drug alone. Roflumilast plus indacaterol combination treatment resulted in significantly elevated phosphorylation of the transcription factor cAMP response element-binding protein (CREB), an effect that was protein kinase A-dependent. Inhibition of protein kinase A was also found to reverse the inhibition of indacaterol and roflumilast on CTGF.ConclusionsThese results demonstrate that addition of roflumilast to a LABA inhibits primary fibroblast/myofibroblast function and therapeutically this may impact lung fibroblast proinflammatory and profibrotic mediator release which contributes to small airway remodeling and airway obstruction in COPD.

The roles of connective tissue growth factor and integrin‐linked kinase in high glucose‐induced phenotypic alterations of podocytes

Emerging evidence has suggested that podocytes undergo epithelial-mesenchymal transition (EMT) in diabetic nephropathy (DN). Connective tissue growth factor (CTGF) and integrin-linked kinase (ILK) are involved in the progression of DN. However, the underlying mechanisms of EMT are not well understood. The study aimed to investigate the roles of CTGF and ILK in high glucose-induced phenotypic alterations of podocytes and determine whether ILK signaling is downstream of CTGF. The epithelial marker of nephrin and the mesenchymal marker of desmin were investigated by real-time RT-PCR and Western blotting. The results demonstrated that podocytes displayed a spreading, arborized morphology in normal glucose, whereas they had a cobblestone morphology in high glucose conditions, accompanied by decreased nephrin expression and increased desmin expression, suggesting podocytes underwent EMT. In response to high glucose, CTGF and ILK expression in podocytes were increased in a dose- and time-dependent manner, whereas the increase did not occur in the osmotic control. Furthermore, the inhibition of CTGF with anti-CTGF antibody prevented the phenotypic transition, as demonstrated by the preservation of epithelial morphology, the suppression of high glucose-induced desmin overexpression and the restoration of nephrin. Of note, the upregulation of ILK induced by high glucose was partially blocked by the inhibition of CTGF. In summary, these findings suggested that CTGF and ILK were involved in high glucose-induced phenotypic alterations of podocytes. ILK acted as a downstream kinase of CTGF and high glucose-induced ILK expression might occur through CTGF-dependent and -independent pathways.

Effect of Rosmarinic Acid on Experimental Diabetic Nephropathy

Connective tissue growth factor (CTGF) plays a pathogenic role in diabetic nephropathy (DN). Rosmarinic acid (RA) is a naturally occurring phenolic acid. This study was conducted to investigate the efficacy of RA on DN and to elucidate the potential mechanism. High glucose (HG)-stimulated cultured human renal proximal tubular epithelial cells (HK-2) analysed CTGF expression by western blotting, and it was investigated whether extracellular signal-regulated kinase (ERK) signalling pathway was involved. Using streptozotocin (STZ)-induced rat animal models, diabetic rats were randomized to receive intragastric (i.g.) doses of RA. Renal tissue, blood and urine samples were collected to determine biochemical index and analyse protein expression. In vitro study, RA reduced CTGF excretion in HG-induced HK-2 cells through the ERK signalling pathway. In an in vivo study, I.g. of RA 7.5 or 15 mg/kg significantly ameliorated renal function and increased body-weight. Meanwhile, RA reduced renal CTGF expression by immunohistochemical staining and reduced serum levels of CTGF. Besides, there were no significant differences in glycaemia levels between the RA groups compared with the STZ-treated group. Furthermore, RA ameliorated renal pathology. These results suggest that RA exerts an early renal protective role to DN. Inhibition of CTGF may be a potential target in DN therapy, which highlights the possibility of using RA in the treatment of DN.

Role of Connective Tissue Growth Factor in the Retinal Vasculature during Development and Ischemia

To investigate the function of connective tissue growth factor (CTGF), a matricellular protein of the CCN (Cyr61/CTGF/Nov) family, in retinal vasculature during development and ischemia. CTGF expression was determined using RT-PCR, immunohistochemistry, and transgenic mice carrying CTGF promoter-driven-GFP. CTGF antibody was intraocularly injected into neonates at postnatal day (P)2, and its effect on retinal angiogenesis was analyzed at P4. Transgenic animals expressing GFP regulated by the glial fibrillary acidic protein promoter were used for astrocyte visualization. Retinal vascular occlusion was introduced by rose Bengal and laser photocoagulation on chimeric mice that were reconstituted with GFP+ bone marrow cells. Vascular repair in response to VEGF-A and CTGF was analyzed. A temporal increase in CTGF at both mRNA and protein levels was observed in the ganglion cell layer and inner nuclear layer during development. Endothelial cells and pericytes were identified as the main cellular sources of CTGF during retinal angiogenesis. CTGF stimulated the migration of astrocytes, retinal endothelial cells, and pericytes in vitro. Inhibition of CTGF by specific antibody affected vascular filopodial extension, growth of the superficial vascular plexus, and astrocyte remodeling. In adult mice, CTGF was prominently expressed in the perivascular cells of arteries. CTGF activated bone marrow-derived perivascular cells and promoted fibrovascular membrane formation in the laser-induced adult retinopathy model. CTGF is expressed in vascular beds and acts on multiple cell types. It is important for vessel growth during early retinal development and promotes the fibrovascular reaction in murine retinal ischemia after laser injury.

Effect of loganin on experimental diabetic nephropathy

Connective tissue growth factor (CTGF) plays a pathogenic role in diabetic nephropathy (DN). Loganin, an iridoid glucoside compound was isolated from Cornus officinalis Sieb. et Zucc. This study was conducted to investigate the efficacy of loganin on DN and to elucidate the potential mechanism. High glucose (HG) stimulated cultured human renal proximal tubular epithelial cells (HK-2) analyzed CTGF expression by Western blotting and investigated whether extracellular signal-regulated kinase (ERK) signaling pathway was involved. Streptozotocin (STZ)-induced experimental DN, randomized to receive intragastric (i.g.) of loganin. Renal tissue, blood and urine samples were collected to determine and analyze. In vitro study, loganin reduced CTGF excretion in HG-induced HK-2 cells through the ERK signaling pathway. In vivo study, I.g. of loganin 5mg/kg or 10mg/kg significantly ameliorated renal function and increased body weight. Meanwhile, loganin reduced renal CTGF expression by immunohistochemical staining, reduced serum levels of CTGF. Besides, there were no significant differences in blood sugar levels between the loganin groups compared to the STZ-treated group. Furthermore, loganin ameliorated renal pathology. These results suggested that loganin exerts an early renal protective role to DN. Inhibition of CTGF may be a potential target in DN therapy, which highlights the possibility of using loganin to treat DN.

Adiponectin reduces connective tissue growth factor in human hepatocytes which is already induced in non-fibrotic non-alcoholic steatohepatitis

Connective tissue growth factor (CTGF) is induced in liver fibrosis and enhances the activity of transforming growth factor β (TGFβ). Recently we have shown that the hepatoprotective adipokine adiponectin downregulates CTGF in primary human hepatocytes (PHH). In the current study, the mechanisms mediating suppression of CTGF by adiponectin and the well described downstream effector of adiponectin receptor 2 (AdipoR2), peroxisome proliferator activated receptor α (PPARα), were analyzed in more detail. Adiponectin downregulated CTGF mRNA and protein in primary human hepatocytes (PHH) and suppression was blocked by a PPARα antagonist indicating that AdipoR2 is involved. The PPARα agonists fenofibrate and WY14643 also reduced CTGF protein in these cells. Adiponectin further impaired TGFβ-mediated upregulation of CTGF. Phosphorylation of the TGFβ downstream effectors SMAD2 and –3 was reduced in PHH incubated with adiponectin or PPARα agonists suggesting that early steps in TGFβ signal transduction are impaired. CTGF and TGFβ mRNA levels were increased in human non-fibrotic non-alcoholic steatohepatitis (NASH), and here AdipoR2 expression was significantly reduced. Current data show that CTGF and TGFβ are already induced in non-fibrotic NASH and this may be partly explained by low adiponectin bioactivity which interferes with TGFβ signaling by reducing phosphorylation of SMAD2/3 and by downregulating CTGF.

Chronic kidney disease growth factors in renal fibrosis

1. The common pathological alteration in virtually every progressive chronic kidney disease (CKD) is renal fibrosis. 2. This review focuses on some growth factors, which are particularly well-established in contributing to fibrosis, such as the profibrotic, transforming growth factor-β, and connective tissue growth factor (CTGF), as well as the antifibrotic, bone morphogenic protein 7. The role of other growth factors is only starting to emerge (e.g. platelet-derived growth factor), and the role of yet others remains unclear (e.g. vascular endothelial growth factor). 3. Whether circulating or excreted, growth factors might serve as biomarkers of renal fibrosis and CKD remains largely unanswered. 4. Animal studies suggest that manipulation of growth factors might be an effective treatment option for patients with renal fibrosis and CKD. So far, only inhibition of CTGF is being tested in patients with diabetic nephropathy.

Increased lysyl oxidase expression and collagen cross-linking during atrial fibrillation

The aim of the study is to characterize the signal transduction leading to interstitial fibrosis in the pathogenesis of atrial fibrillation (AF) and atrial remodeling. Samples of the left atrial appendage (LA) from patients with AF showed higher collagen content (73 ± 5 vs. 38 ± 2 μg/mg protein) and 2.5-fold increased collagen crosslinking compared to patients with sinus rhythm (SR). Affymetrix-assays, RT-PCR and western Blot analysis revealed that LA of AF patients are characterized by increased lysyl oxidase (LOX) mRNA (218 ± 42%) and protein (253 ± 11%) expression. This was associated with increased expression of connective tissue growth factor (CTGF), fibronectin and Rac1 activity compared to SR. In neonatal cardiac fibroblasts, the Rac1 specific small molecule inhibitor NSC23766 prevented angiotensin II (AngII) induced upregulation of LOX (214 ± 16%) expression. Inhibition of CTGF by siRNA transfections completely inhibited AngII induced LOX expression. The LOX specific small molecule inhibitor BAPN prevented AngII and CTGF induced fibronectin expression. Left atria of transgenic mice with cardiac overexpression of Rac1 (RacET) that develop AF at high age exhibited upregulation of CTGF as well as LOX (187 ± 7%) and fibronectin (627 ± 146%) expression. Atria of RacET showed increased collagen content (28 ± 2 μg/mg protein) and crosslinking (10 ± 0.7) compared to wildtypes (20 ± 0.4 μg/mg protein; 5 ± 0.9). Left atrial myocardium of patients with atrial fibrillation is characterized by increased lysyl oxidase and fibronectin expression as well as collagen cross-linking. In cardiac fibroblasts, Rac1 GTPase mediates upregulation of fibronectin via LOX and CTGF. Inhibition of this signaling pathway may therefore represent a target for the prevention of fibrotic atrial remodeling.

The cannabinoid WIN55, 212-2 abrogates dermal fibrosis in scleroderma bleomycin model

ObjectivesThere is increasing evidence that the endocannabinoid system may be involved in pathological fibrosis, and that its modulation might limit fibrotic responses. The aim of this study was to examine...

Connective Tissue Growth Factor (CTGF) Is Essential for Self Renewal and Proliferation of Mesenchymal Stromal Cells (MSCs) and Affects Leukemia-Stromal Interactions.

AbstractAbstract 3845Connective tissue growth factor (CTGF) is a member of the CCN family of growth factors that are critical regulators of vertebrate development. CTGF is a secreted protein that promotes extracellular matrix production, chemotaxis, cell proliferation and integrin expression. Since CTGF is highly expressed in acute lymphoblastic leukemia (ALL) and CTGF expression levels are related to ALL patient survival (Olga ST et al., Blood, 2007), we hypothesized that CTGF plays a role in regulating stromal cell proliferation and leukemia-stroma interaction. Our first goal was to characterize multipotent mesenchymal stromal cells (MSCs) from CTGF-/- mice, which die soon after birth from respiratory failure due to abnormal skeletal growth. We first attempted to isolate MSCs from BALB/C wild type and CTGF-/- newborn mice (Lvkovic S et al., Development, 2003). While we had no difficulty isolating MSCs from wild type mice, we failed to generate MSCs from CTGF-/- newborns (bone marrow, liver, spleen and thymus). As an alternative approach, we suppressed CTGF in bone marrow derived human MSCs with lenti-virus delivered CTGF shRNA (CTGF-KD-MSCs) and achieved knockdown of ≂f65% compared to vector control cells as determined by RT-PCR. Shortly after transduction (4 days) we started to observe major changes in the phenotype of CTGF-KD-MSCs, which grew 6–7 fold slower compared to vector control MSCs. CTGF-KD-MSCs displayed a significant decrease in the number of cells in S phase (from 14.7% ± 0.8% to 3.5%± 0.4%) and a concomitant increase in the number of G0/1 cells (from 68.8%±1.8% to 82.4%± 1.3%) suggesting a G0 or G1 block. To investigate if CTGF affects expression of MSC surface proteins, we analyzed standard MSC markers including CD105, CD90, CD73, CD44, CD140b, CD166 and CD45 (as a negative marker). Surprisingly, no differences in the expression of these markers in CTGF-KD-MSCs compared to vector control MSCs were observed. To determine if CTGF might regulate gene expression in MSCs, we performed a microarray analysis using Illumina arrays. By gene set analysis methods, we observed significant down regulation (p < 10-9 by the hypergeometric distribution test) in CTGF-KD-MSCs of genes involved in cell cycle progression, most notably in the Gene Ontology lists for ribosomal biogenesis and mitosis. As MSCs are known to differentiate into mesodermal cell lineages, we next tested the differentiation potential of CTGF-KD-MSCs as compared to the vector control MSCs. Following standard differentiation protocols, both CTGF-KD- and vector control-MSCs differentiated into osteoblasts and chondrocytes with no differences in their ability to differentiate into these lineages; whereas, CTGF-KD-MSCs showed 6–7 fold increase in their adipocyte differentiation potential compared to vector control MSCs. Another property of MSCs is to self-renew and generate colony forming units-fibroblast like (CFU-F). Fifty or 100 cells of either CTGF-KD-MSCs or vector control MSCs were seeded in alpha-MEM with 10% serum. After 2 weeks of culture in alpha-MEM with 10% serum, the resulting fibroblast-like colonies were counted. Vector control MSCs generated ≂f30-fold higher CFU-F compared to CTGF-KD-MSCs indicating that CTGF-KD-MSCs lack the ability to self renew. Next we determined if suppression of CTGF might impede leukemia cell migration to MSCs. Using standard trans-well assays, we examined human pre-B acute lymphoblastic leukemia (ALL) derived REH cells and found that they migrated 45% ± 4% less to CTGF-KD-MSCs compared to control MSCs. These findings suggest that CTGF plays a major role in stromal cell proliferation, self renewal and adipocyte differentiation of MSCs. In addition, CTGF is involved in leukemia cell migration towards MSCs and inhibition of CTGF impairs the migration of leukemic cells towards stromal cells and thereby provides opportunities to prevent homing of leukemic cells and sensitize them to chemotherapy. Disclosures:Andreeff:MyeloRx LLC: Consultancy, participant in research under an NCI SBIR Contract to MyeloRx LLC.

Attenuation and Reversal of Radiation-induced Pulmonary Fibrosis in a Murine Model by an Anti-CTGF Monoclonal Antibody

Purpose/Objective(s): Connective tissue growth factor (CTGF) is a matricellular protein that is a central mediator of tissue remodeling. CTGF expression is elevated in patients with idiopathic pulmonary fibrosis. Several studies in different organs have suggested that CTGF is essential for sustained fibrosis. Therefore, inhibition of CTGF may be therapeutically beneficial to fibrosis patients. Radiation induced lung fibrosis is a dose limiting side effect. Here we investigated this concept in a murine model of radiation induced lung fibrosis.

Targeted inhibition of CTGF by RNA interference restrain renal fibrosis

Objective To investigate the impact on renal fibrosis by inhibition of connective tissue growth factor( CTGF) by RNA interference in spontaneous hypertension rat( SHR) . Method Twenty SHR were randomly (random number) divided into SHR group ( n = 10) and RNAi group ( n = 10), eight Wistar-Kyoto rats were set as control. At the end of RNA interference procedure, all the rats were sacrificed and the kidneys were harvested. The mRNA and plasmosin of CTGF and fibronectin(FN) of renal tissue were extracted and measured by RT-PCR and Western Blotting. And the localization of CTGF and FN were analyzed with immunohistochernistry technique. The collagen deposition(shown as collagen volume traction, CVF) were evaluated with 0.1% sirius-picric staining, and the hydroxyproline of myocardium were detected by colorimetry. Results The mRNA and protein expression of CTGF decreased 66% and 62% in RNAi group (P < 0.01). The mRNA and protein expression of FN decreased 56% and 51% in RNAi group.The same inhibition effect was observed by hislological analysis. Immuno-histochemistry showed that CTGF localized both in renal parenchyma and renal interstitium, whereas FN majorly expressed in renal interstitium. Observation with light microscope showed that collagen deposition(CVF)decreased sharply in RNAi group versus SHR group. And the same effect was viewed in hydroxypnoline assay[SHR group: (0.596 ± 0.067) μg/mg, RNAi group: (0.368±0.084) μg/mg, P < 0.01 ] .Further study by polarized microscope displayed that RNA interference mainly suppressed type I collagen synthesis. Conclusions Targeted inhibition of CTGF by RNA interference leads significant decrease of extracellular matrix deposition in kidney. And the anti-fibrotic effect independent of lower the blood pressure. This study indicated CTGF take a key role in the development and progress of renal fibrosis. Key words: Connective tissue growth factor; Renal fibrosis; RNA interference; Extracellular matrix; Fi-bronectin; Collagen; Plasmid; Transfection

Effect of Astilbin on Experimental Diabetic Nephropathyin vivoandin vitro

Astilbin, a flavonoid compound, was isolated from the rhizome of Smilax glabra Roxb. This study was conducted to investigate the efficacy of astilbin on experimental diabetic nephropathy (DN) in vivo and in vitro and its possible mechanisms. Astilbin was added in high glucose stimulated HK-2 cells, streptozotocin-induced experimental DN, randomized to receive intragastric ( I. G.) astilbin to observe its anti-renal lesion effect. Results showed that astilbin inhibited high glucose stimulated HK-2 cell production of transforming growth factor-beta1 (TGF-beta1) and connective tissue growth factor (CTGF) in vitro, especially CTGF; analogic results was also found in vivo. I. G. of astilbin 2.5 mg/kg or 5 mg/kg significantly ameliorated renal function, reduced kidney index, while it increased body weight and survival time in animals. In addition there was no significant difference in blood glucose level between the STZ-treated group and the astilbin groups. Furthermore, astilbin ameliorated the pathological progress of renal morphology. Astilbin can exert an early renal protective role to DN, inhibit production of TGF-beta1 and especially of CTGF. We suggest that astilbin inhibition of CTGF may be a potential target in DN therapy. This work provides the first evidence for astilbin as a new candidate of DN therapeutic medicine.