Romana Schäfer

Colony-Stimulating Factor-1 Blockade by Antisense Oligonucleotides and Small Interfering RNAs Suppresses Growth of Human Mammary Tumor Xenografts in Mice

Colony-stimulating factor (CSF)-1 is the primary regulator of tissue macrophage production. CSF-1 expression is correlated with poor prognosis in breast cancer and is believed to enhance mammary tumor progression and metastasis through the recruitment and regulation of tumor-associated macrophages. Macrophages produce matrix metalloproteases (MMPs) and vascular endothelial growth factor, which are crucial for tumor invasion and angiogenesis. Given the important role of CSF-1, we hypothesized that blockade of CSF-1 or the CSF-1 receptor (the product of the c-fms proto-oncogene) would suppress macrophage infiltration and mammary tumor growth. Human MCF-7 mammary carcinoma cell xenografts in mice were treated with either mouse CSF-1 antisense oligonucleotide for 2 weeks or five intratumoral injections of either CSF-1 small interfering RNAs or c-fms small interfering RNAs. These treatments suppressed mammary tumor growth by 50%, 45%, and 40%, respectively, and selectively down-regulated target protein expression in tumor lysates. Host macrophage infiltration; host MMP-12, MMP-2, and vascular endothelial growth factor A expression; and endothelial cell proliferation within tumors of treated mice were decreased compared with tumors in control mice. In addition, mouse survival significantly increased after CSF-1 blockade. These studies demonstrate that CSF-1 and CSF-1 receptor are potential therapeutic targets for the treatment of mammary cancer.

Colony-Stimulating Factor-1 Antibody Reverses Chemoresistance in Human MCF-7 Breast Cancer Xenografts

Overexpression of colony-stimulating factor-1 (CSF-1) and its receptor in breast cancer is correlated with poor prognosis. Based on the hypothesis that blockade of CSF-1 would be beneficial in breast cancer treatment, we developed a murinized, polyethylene glycol-linked antigen-binding fragment (Fab) against mouse (host) CSF-1 (anti-CSF-1 Fab). Mice bearing human, chemoresistant MCF-7 breast cancer xenografts were treated with combination chemotherapy (CMF: cyclophosphamide, methotrexate, 5-fluorouracil; cycled twice i.p.), anti-CSF-1 Fab (i.p., cycled every 3 days for 14 days), combined CMF and anti-CSF-1 Fab, or with Ringers solution as a control. Anti-CSF-1 Fab alone suppressed tissue CSF-1 and retarded tumor growth by 40%. Importantly, in combination with CMF, anti-CSF-1 Fab reversed chemoresistance of MCF-7 xenografts, suppressing tumor development by 56%, down-regulating expression of the chemoresistance genes breast cancer-related protein, multidrug resistance gene 1, and glucosylceramide synthase, and prolonging survival significantly. Combined treatment also reduced angiogenesis and macrophage recruitment and down-regulated tumor matrix metalloproteinase-2 (MMP-2) and MMP-12 expression. These studies support the paradigm of CSF-1 blockade in the treatment of solid tumors and show that anti-CSF-1 antibodies are potential therapeutic agents for the treatment of mammary cancer.

Selective downregulation of VEGF-A(165), VEGF-R(1), and decreased capillary density in patients with dilative but not ischemic cardiomyopathy.

Cardiomyopathy (CM) comprises a heterogeneous group of diseases, including ischemic (ICM) and dilative (DCM) forms. The pathogenesis of primary DCM is not clearly understood. Recent studies in mice show that vascular endothelial growth factor (VEGF) is involved in ICM. Whether VEGF plays a role in human CM is unknown. We examined the mRNA and protein expression of VEGF and its receptors in hearts of patients with end-stage DCM and ICM and in healthy individuals using real-time polymerase chain reaction and Western blotting. Number of capillaries, area of myocytes, and collagen were calculated in cardiac biopsies using transmission electron microscopy. In DCM, except for VEGF-C, mRNA transcript levels of VEGF-A165, VEGF-A189, and VEGF-B and the protein level of VEGF-A and VEGF-R1 were downregulated compared with controls (P <0.05). However, in ICM, mRNA transcript levels of VEGF isoforms and protein levels of VEGF-C were upregulated. The vascular density was decreased in DCM but increased in ICM compared with controls (P <0.05). Muscular hypertrophy was not different for ICM and DCM, although DCM had more collagen (P <0.05). Blunted VEGF-A and VEGF-R1 protein expression and downregulated mRNA of the predominant isoform of VEGF-A, VEGF-A165, to our knowledge shown here for the first time, provide evidence that the VEGF-A defect in DCM is located upstream. Whether downregulation of certain VEGF isoforms in DCM is a cause or consequence of this disorder remains unclear, although upregulated VEGF levels in ICM are most likely the result of ischemia.

Fibrin-embedded administration of VEGF plasmid enhances skin flap survival.

The aim of the present study was to experimentally evaluate whether topical fibrin‐mediated administration of a vascular endothelial growth factor (VEGF)‐A plasmid to the wound bed can protect skin flaps from necrosis. A plasmid expression vector containing the VEGF‐A cDNA was constructed. The plasmid was then administered to the wound bed of rat abdominal skin flaps in a fibrin sealant. The percentage of viable, ischemic and necrotic tissue was assessed postoperatively as a baseline and after 3 and 7 days using digital surface area morphometry. Laser Doppler imaging of the flaps and VEGF‐A Western blot analysis of flap tissue were performed to assess angiogenesis and VEGF‐A tissue levels. Flaps treated with VEGF plasmids in the presence of uptake enhancing Lipofectamine transfection reagent increased flap survival 7 days postoperatively significantly associated with markedly elevated tissue perfusion and enhanced tissue VEGF‐A protein expression. Our results indicate that topical fibrin‐mediated administration of a VEGF‐A plasmid may serve as an alternative to previous strategies in treating ischemic skin flaps. The suggested therapeutic approach is easily applicable and inexpensive in preparation. Thus, this protocol may also enhance wound healing in posttrauma skin lacerations or in skin grafts.

Impaired VE-Cadherin/β-Catenin Expression Mediates Endothelial Cell Degeneration in Dilated Cardiomyopathy

Background—The cross-talk between vascular endothelial growth factor (VEGF)-A, angiopoietin (Ang), and VE-cadherin coregulates endothelial cell (EC) survival. Cardiac expression of VEGF-A but not its receptor KDR is blunted in dilated cardiomyopathy (DCM). Whether VE-cadherin/Ang function is affected in DCM is unknown. Methods and Results—The myocardial expression of VE-cadherin/&bgr;-catenin, Ang-1, Ang-2, and their receptor Tie-2 was examined in DCM, ischemic cardiomyopathy (ICM), and in control subjects through the use of real-time RT-PCR, Western blotting, and immunocytochemistry. EC degeneration was quantified by TEM. RNA interference against VE-cadherin and VEGF deprivation and stimulation were applied to cultured DCM myocardium and human microvascular ECs to examine the interplay between VEGF, VE-cadherin/&bgr;-catenin, and Ang-2. Analysis of tissue sections with similar rates of EC degeneration in both patient groups showed that VE-cadherin/&bgr;-catenin expression was downregulated in DCM only (P <0.05). Although Ang-1 was not changed, Ang-2 expression was downregulated and Tie-2 protein expression was upregulated both in DCM and ICM (P <0.05). The ratio of degenerated to normal ECs was significantly higher in DCM versus ICM (P <0.05). Targeted VE-cadherin gene silencing in cultured human ECs resulted in similar degenerative effects observed in myocardial ECs of DCM patients. In vitro experiments indicated that VE-cadherin/&bgr;-catenin expression is independent of VEGF. Conclusions—These results indicate for the first time that the EC survival is impaired in myocardium of patients with DCM involving VE-cadherin/&bgr;-catenin, probably independent of VEGF. Targeting VE-cadherin might be of benefit to counteract the selective EC pathology in DCM.

Stromal cell-derived CSF-1 blockade prolongs xenograft survival of CSF-1-negative neuroblastoma

The molecular mechanisms of tumor–host interactions that render neuroblastoma (NB) cells highly invasive are unclear. Cancer cells upregulate host stromal cell colony‐stimulating factor‐1 (CSF‐1) production to recruit tumor‐associated macrophages (TAMs) and accelerate tumor growth by affecting extracellular matrix remodeling and angiogenesis. By coculturing NB with stromal cells in vitro, we showed the importance of host CSF‐1 expression for macrophage recruitment to NB cells. To examine this interaction in NB in vivo, mice bearing human CSF‐1‐expressing SK‐N‐AS and CSF‐1‐negative SK‐N‐DZ NB xenografts were treated with intratumoral injections of small interfering RNAs directed against mouse CSF‐1. Significant suppression of both SK‐N‐AS and SK‐N‐DZ NB growth by these treatments was associated with decreased TAM infiltration, matrix metalloprotease (MMP)‐12 levels and angiogenesis compared to controls, while expression of tissue inhibitors of MMPs increased following mouse CSF‐1 blockade. Furthermore, Tie‐2‐positive and ‐negative TAMs recruited by host CSF‐1 were identified in NB tumor tissue by confocal microscopy and flow cytometry. However, host‐CSF‐1 blockade prolonged survival only in CSF‐1‐negative SK‐N‐DZ NB. These studies demonstrated that increased CSF‐1 production by host cells enhances TAM recruitment and NB growth and that the CSF‐1 phenotype of NB tumor cells adversely affects survival.

Differential role of TGF-beta1/bFGF and ET-1 in graft fibrosis in heart failure patients.

Collagen overproduction characteristic for dilated cardiomyopathy (DCM) is coregulated by endothelin (ET)‐1, transforming growth factor (TGF)‐β1, basic fibroblast growth factor (bFGF) and matrix metalloproteases (MMPs). Whether these molecules affect grafts transplanted to heart failure patients is unknown. In 67 idiopathic DCM patients, 31 patients with ischemic cardiomyopathy (ICM) and 16 controls, the myocardial bFGF, TGF‐β1, pro‐collagen (PrCol) type 1 (PrCol1‐α1, ‐α2) and MMP expressions were examined using real‐time RT‐PCR or Western blotting. mRNA expression was measured in grafts for 1 year. TGF‐β1/bFGF stimulation or gene silencing was used to examine their effect on collagen synthesis in cardiac tissue cultures. TGF‐β1 and PrCol1 were upregulated in DCM only, while bFGF was upregulated in both groups versus controls. TGF‐β1 downregulated MMP‐1 and upregulated collagen 1, whereas bFGF upregulated MMP‐13 in DCM tissue. Post‐transplant PrCol1‐α1, ‐α2 and ET‐1 mRNA increased over time in grafts of DCM patients only, while other factors returned to control baseline levels in DCM and ICM. These data indicate that cardiac transplantation corrects the dysregulated TGF/bFGF/MMP‐1/MMP‐13, but not the excess collagen and ET‐1 synthesis in cardiac grafts transplanted to DCM patients. ET‐1 might be a major pathologic trigger for graft fibrosis in DCM.

Co-expression of endothelin-1 and vascular endothelial growth factor mediates increased vascular permeability in lung grafts before reperfusion

Endothelin-1 (ET-1) protein levels increase in lung transplant recipients, although the source of ET-1 remains uncertain. Evidence is accumulating that ET-1 coregulates the expression of vascular endothelial growth/permeability factor (VEGF-A). By using real-time polymerase chain reaction and Western blotting, we describe upregulated ET-1 mRNA and VEGF-A protein levels as well as increased fluid content in donor lung-tissue biopsies before reperfusion. Hypoxia and ET-1 co-upregulate VEGF-A overexpression, therefore temporary VEGF-A antagonism during graft preservation might be of benefit in lung transplantation.

Donor Myocardial HIF-1α Is an Independent Predictor of Cardiac Allograft Dysfunction: A 7-Year Prospective, Exploratory Study

Knowledge on interplay between the cardiac molecular response to transplantation‐induced stress and primary graft dysfunction (PGD) is limited. A cDNA array identified HIF‐1, EGR‐1, NAB‐2, VEGF‐A and uPA as mediators of cardiac tissue response to transplantation‐induced stress. mRNA expression of these molecules was measured in left ventricular biopsies from 200 donors before and after aortic cross‐clamping and at 10‐, 30‐ and 60‐min reperfusion by real‐time RT‐PCR. HIF‐1α expression at two time points was significantly associated with PGD, as shown by univariate analysis, receiver operating characteristic curve and multivariate logistic regression. At a cut‐off level of 200 arbitrary units, HIF‐1α after aortic cross‐clamping in donors (78% sensitivity, 83% specificity) and at 10‐min reperfusion (85% sensitivity, 83% specificity) identified PGD. HIF‐1α demonstrates the potential to be a predictive marker for PGD; however, as multiple factors were tested at different time points, prospective evaluation is clearly necessary to confirm this observation.

Serum Matrix Metalloprotease‐1 and Vascular Endothelial Growth Factor–A Predict Cardiac Allograft Rejection

Cardiac allograft rejection is currently diagnosed from endomyocardial biopsies (EMB) that are invasive and impractical to repeat. A serological marker could facilitate rejection monitoring and minimize EMB‐associated risks. We investigated the relation of serum matrix metalloprotease (MMP)‐1 and vascular endothelial growth factor (VEGF)‐A concentrations to cardiac allograft rejection, using 1176 EMBs and serum samples obtained from 208 recipients. Acute cellular rejection was diagnosed in 186 EMBs. Mean week 1 and week 2 serum MMP‐1 concentrations predicted rejection (p = 0.001, AUC = 0.80). At the optimal cut‐off level of ≥7.5 ng/mL, MMP‐1 predicted rejection with 82% sensitivity and 72% specificity. Initial serum MMP‐1 <5.3 ng/mL (lowest quartile) was associated with rejection‐free outcome in 80% of patients. Both MMP‐1 (p < 0.001, AUC = 0.67–0.75) and VEGF‐A (p < 0.01, AUC = 0.62–0.67) predicted rejection on the next EMB, while rejection at EMB was identified only by VEGF‐A (p < 0.02, AUC = 0.70–0.77). Patients receiving combined cyclosporine‐A and everolimus had the lowest serum MMP‐1 concentrations. While serum MMP‐1 predicts rejection‐free outcome and VEGF‐A identifies rejection on EMB, both markers predict rejection in follow‐up of cardiac transplant recipients. Combination of serum MMP‐1 and VEGF‐A concentration may be a noninvasive prognostic marker of cardiac allograft rejection, and could have important implications for choice of surveillance and immunosuppression protocols.