Single-dose GSTP1 prevents infarction-induced heart failure.

BACKGROUND: Heart failure (HF) is a common and often fatal complication of myocardial infarction (MI). Glutathione S-transferase P1-1 (GSTP1) has antiapoptotic and antiinflammatory effects and is a specific serum marker in HF patients. However, its role in HF treatment is unknown. METHODS AND RESULTS: GSTP1 effect was examined in a rat MI-induced HF model. Magnetic resonance imaging was used to examine cardiac function. GSTP1 and tumor necrosis factor α receptor-associated factor 2 (TRAF2) mRNA and protein expression were elevated in failing myocardium, although GSTP-1 binding activity to TRAF2 was not changed versus control. HF was associated with higher active JNK1 and p38 protein expression but reduced GSTP-1 binding activity to JNK1 and p38. Recombinant GSTP1 inhibited JNK1 and p38 and enhanced its own binding activity to TRAF2 and JNK1 in vitro. In the HF model, single-dose GSTP1 treatment reduced infarct area, apoptosis, and the expression of JNK1, p38, nuclear factor κB, and proinflammatory cytokines and improved thinning ratio, cardiac index and output, stroke volume, ejection fraction, regional wall motion, and survival compared with control. CONCLUSIONS: GSTP1 application early after MI results in long-term beneficial structural and functional effects that prevent progression to HF. GSTP1 could be a novel adjunct myocardial salvage approach in patients after MI.

Egr-1 upregulates Siva-1 expression and induces cardiac fibroblast apoptosis.

The early growth response transcription factor Egr-1 controls cell specific responses to proliferation, differentiation and apoptosis. Expression of Egr-1 and downstream transcription is closely controlled and cell specific upregulation induced by processes such as hypoxia and ischemia has been previously linked to multiple aspects of cardiovascular injury. In this study, we showed constitutive expression of Egr-1 in cultured human ventricular cardiac fibroblasts, used adenoviral mediated gene transfer to study the effects of continuous Egr-1 overexpression and studied downstream transcription by Western blotting, immunohistochemistry and siRNA transfection. Apoptosis was assessed by fluorescence microscopy and flow cytometry in the presence of caspase inhibitors. Overexpression of Egr-1 directly induced apoptosis associated with caspase activation in human cardiac fibroblast cultures in vitro assessed by fluorescence microscopy and flow cytometry. Apoptotic induction was associated with a caspase activation associated loss of mitochondrial membrane potential and transient downstream transcriptional up-regulation of the pro-apoptotic gene product Siva-1. Suppression of Siva-1 induction by siRNA partially reversed Egr-1 mediated loss of cell viability. These findings suggest a previously unknown role for Egr-1 and transcriptional regulation of Siva-1 in the control of cardiac accessory cell death.

Targeting Cdc42 with the small molecule drug AZA197 suppresses primary colon cancer growth and prolongs survival in a preclinical mouse xenograft model by downregulation of PAK1 activity.

BACKGROUND: Rho GTPases play important roles in cytoskeleton organization, cell cycle progression and are key regulators of tumor progression. Strategies to modulate increased Rho GTPase activities during cancer progression could have therapeutic potential. METHODS: We report here the characterization of a Cdc42-selective small-molecule inhibitor AZA197 for the treatment of colon cancer that was developed based on structural information known from previously developed compounds affecting Rho GTPase activation. We investigated the effects of AZA197 treatment on RhoA, Rac1 and Cdc42 activities and associated molecular mechanisms in colon cancer cells in vitro. Therapeutic effects of AZA197 were examined in vivo using a xenograft mouse model of SW620 human colon cancer cells. After treatment, tumors were excised and processed for Ki-67 staining, TUNEL assays and Western blotting to evaluate proliferative and apoptotic effects induced by AZA197. RESULTS: In SW620 and HT-29 human colon cancer cells, AZA197 demonstrated selectivity for Cdc42 without inhibition of Rac1 or RhoA GTPases from the same family. AZA197 suppressed colon cancer cell proliferation, cell migration and invasion and increased apoptosis associated with down-regulation of the PAK1 and ERK signaling pathways in vitro. Furthermore, systemic AZA197 treatment reduced tumor growth in vivo and significantly increased mouse survival in SW620 tumor xenografts. Ki-67 staining and tissue TUNEL assays showed that both inhibition of cell proliferation and induction of apoptosis associated with reduced PAK/ERK activation contributed to the AZA197-induced therapeutic effects in vivo. CONCLUSIONS: These data indicate the therapeutic potential of the small-molecule inhibitor AZA197 based on targeting Cdc42 GTPase activity to modulate colorectal cancer growth.

Innovative, simplified orthotopic lung transplantation in rats.

BACKGROUND: Various techniques of orthotopic single lung transplantation in rats have been reported; however, their widespread use has been limited owing to the complexity of the procedure. We report a novel microsurgical lung transplantation model in rats with a high survival rate that can be performed by one surgeon alone. METHODS: A total of 90 left lung allografts were transplanted from Fischer to Wistar Kyoto rats. We developed a triple axis precision system to place and stabilize the vascular clips intrathoracically to clamp the bronchovascular structures, thereby avoiding interference with the heart and contralateral lung movement. A single-suture bronchial anastomosis technique and proximal cuffing approach for vascular anastomosis was used, rendering surgical assistance unnecessary. RESULTS: In our recent series, both short-term (12 h) and long-term (21 d) survival was 100%. The lungs showed excellent perfusion and ventilation immediately on transplantation. Blood gas samples drawn from the left pulmonary vein and the histologic sections revealed excellent graft function. The donor operation lasted 20 ± 2 min, donor left lung dissection required 20 ± 2 min, and implantation required 90 ± 5 min. CONCLUSIONS: The present innovative method of left orthotopic single lung transplantation can be performed by one experienced surgeon alone, with excellent results and a high degree of reproducibility.

Inhibition of stromal PlGF suppresses the growth of prostate cancer xenografts.

The growth and vascularization of prostate cancer is dependent on interactions between cancer cells and supporting stromal cells. The primary stromal cell type found in prostate tumors is the carcinoma-associated fibroblast, which produces placental growth factor (PlGF). PlGF is a member of the vascular endothelial growth factor (VEGF) family of angiogenic molecules and PlGF mRNA levels increase after androgen deprivation therapy in prostate cancer. In this study, we show that PlGF has a direct dose-dependent proliferative effect on human PC-3 prostate cancer cells in vitro and fibroblast-derived PlGF increases PC-3 proliferation in co-culture. In xenograft tumor models, intratumoral administration of murine PlGF siRNA reduced stromal-derived PlGF expression, reduced tumor burden and decreased the number of Ki-67 positive proliferating cells associated with reduced vascular density. These data show that targeting stromal PlGF expression may represent a therapeutic target for the treatment of prostate cancer.

Serum glutathione S-transferase P1 1 in prediction of cardiac function.

BACKGROUND: Glutathione S-transferase P1 1 (GSTP1) belongs to the multigene isozyme family involved in cellular response to oxidative stress and apoptosis. Our initial retrospective proteomic analysis suggested that GSTP1 is associated with heart failure (HF). Although pro-B-type natriuretic peptide (proBNP) serves currently as a surrogate diagnostic and prognostic parameter in HF patients, its specificity remains uncertain. We hypothesized that GSTP1 might be a useful serum marker in the monitoring of HF patients. METHODS AND RESULTS: Serum GSTP1 and proBNP were prospectively measured in 193 patients subdivided based on their ejection fraction (EF) either in equal-sized quintiles or predefined EF groups >52%, 43%-52%, 33%-42%, 23%-32% and ≤22%. At a cutoff of ≥231 ng/mL, GSTP1 identified HF patients with EF ≤22% with 81% sensitivity and 83% specificity, and at a cutoff of ≥655 pg/mL, proBNP identified the same patient group with 84% sensitivity and 22% specificity. GSTP1 at a ≥126 ng/mL cutoff identified EF ≤42% with 90% sensitivity and 95% specificity, or proBNP at a ≥396 pg/mL cutoff had 97% sensitivity and 20% specificity. In regression analyses, GSTP1, but not proBNP, discriminated between EF ≤42% and EF >42% in HF patients. CONCLUSIONS: These results suggest that GSTP1 is strongly associated with HF and could serve as a sensitive and specific marker to predict the ventricular function in HF patients.

The influence of low molecular weight heparin on the expression of osteogenic growth factors in human fracture healing.

PURPOSE: Anticoagulant therapy with low molecular weight heparins (LMWH) and mechanical compression is considered the gold standard for the prevention of thrombosis. However, evidence exists that LMWHs impair bone metabolism. The aim of this study was therefore to analyse alterations in the expression of M-CSF, VEGF and TGF-ß1 after treatment with enoxaparin in patients with long bone fracture to investigate the effect of LMWH on human fracture healing. METHODS: A total of 81 patients with long bone fractures were included in the study and divided into two groups. One group comprised patients who received enoxaparin and the other group, patients who did not receive enoxaparin postoperatively. Growth factor levels were analysed in patients' serum and different groups were retrospectively compared. RESULTS: M-CSF serum concentrations were found to be significantly higher only at 48 weeks after surgery in enoxaparin. Mean overall VEGF serum concentration was higher in patients with enoxaparin. TGF-ß1 serum concentrations were higher at 48 weeks after surgery in patients with enoxaparin. CONCLUSION: This is the first comparative systemic measurement of M-CSF, VEGF and TGF-ß1 serum levels in patients with and without enoxaparin after long bone fracture. Significant differences of the expression of the growth factors after enoxaparin therapy were only observed at week 48 after surgery for M-CSF and TGF-ß1.

The influence of M-CSF on fracture healing in a mouse model.

Macrophage colony-stimulating factor 1 (M-CSF) is known to play a critical role during fracture repair e.g. by recruiting stem cells to the fracture site and impacting hard callus formation by stimulating osteoclastogenesis. The aim of this experiment was to study the impact of systemic M-CSF application and its effect on bony healing in a mouse model of femoral osteotomy. Doing so, we studied 61 wild type (wt) mice (18-week-old female C57BL/6) which were divided into three groups: (1) femoral osteotomy, (2) femoral osteotomy + stabilization with external fixator and (3) femoral osteotomy + stabilization with external fixator + systemic M-CSF application. Further, 12 op/op mice underwent femoral osteotomy and served as proof of concept. After being sacrificed at 28 days bony bridging was evaluated ex vivo with µCT, histological and biomechanical testing. Systemic M-CSF application impacted osteoclasts numbers, which were almost as low as found in op/op mice. Regarding callus size, the application of M-CSF in wt mice resulted in significantly larger calluses compared to wt mice without systemic M-CSF treatment. We further observed an anabolic effect of M-CSF application resulting in increased trabecular thickness compared to wt animals without additional M-CSF application. Systemic M-CSF application did not alter biomechanical properties in WT mice. The impact of M-CSF application in a mouse model of femoral osteotomy was oppositional to what we were expecting. While M-CSF application had a distinct anabolic effect on callus size as well as trabecular thickness, this on bottom line did not improve biomechanical properties. We hypothesize that in addition to the well-recognized negative effects of M-CSF on osteoclast numbers this seems to further downstream cause a lack of feedback on osteoblasts. Ultimately, continuous M-CSF application in the absence of co-stimulatory signals (e.g. RANKL) might overstimulate the hematopoietic linage in favor of tissue macrophages instead of osteoclasts.

Donor serum SMARCAL1 concentrations predict primary graft dysfunction in cardiac transplantation.

BACKGROUND: Primary graft dysfunction (PGD) is a life-threatening complication in cardiac transplantation. A sensitive, specific, and easily measurable predictor in donors could facilitate PGD prevention. METHODS AND RESULTS: SMARCAL1 is a matrix-associated regulator of chromatin with helicase and ATPase activities, and its serum concentrations were significantly increased in a targeted protein array in donors whose grafts developed PGD. Therefore, this study analyzed SMARCAL1 serum concentrations by ELISA in 336 heart donors before and after aortic cross-clamping (ACC) and in recipients at 10, 30, and 60 minutes reperfusion. Demographic and hemodynamic parameters of donors and recipients as well as transplant procedure characteristics were documented. PGD (n=68) was defined as ventricular dilation and hypocontractility associated with systolic blood pressure <90 mm Hg, pulmonary capillary wedge pressure >20 mm Hg, and decreased mixed venous oxygen saturation necessitating mechanical circulatory support. SMARCAL1 serum protein concentration was significantly increased only before and after ACC in donors (P<0.0001) whose grafts developed PGD compared to those who did not. In receiver operating characteristic curve analysis, SMARCAL1 serum concentration at a cut-off level of > or =1.25 ng/mL before ACC in donors predicted PGD (P<0.0001, AUC=0.988, OR=17.050, 95% CI=5.200 to 55.901) with 96% sensitivity and 88% specificity. SMARCAL1 serum concentrations <1.25 ng/mL in donors before ACC resulted in 97% PGD-free outcome and SMARCAL1 concentrations > or =1.25 resulted in 83% PGD occurrence. CONCLUSIONS: Donor serum SMARCAL1 may serve as a specific, sensitive, and noninvasive predictive marker in the assessment of cardiac graft quality.

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.

Adenoviral-mediated endothelial precursor cell delivery of soluble CD115 suppresses human prostate cancer xenograft growth in mice.

Prostate cancer tumor growth and neovascularization is promoted by an interplay between migratory tumor stromal cells such as specialized tumor-associated macrophages (TAMs) and circulating endothelial precursor cells (CEPs). As vehicles for tumor therapy, human CEPs are relatively easy to isolate from peripheral blood, are able to proliferate long-term in vitro, are amenable to viral manipulation, and preferentially home to regions of ischemia found in growing tumors. We show here that human peripheral blood CEPs expanded ex vivo migrate to prostate cancer cells in vitro and efficiently home to human prostate tumor xenografts in vivo. Infection of precursors ex vivo with an adenovirus constructed to secrete a soluble form of the colony-stimulating factor-1 receptor CD115 that inhibits macrophage viability and migration in vitro significantly decreases the number of TAMs in xenografts (p < .05), reduces proliferation (p < .01) and vascular density (p < .03), and suppresses the growth of xenografts (p < .03). These data show for the first time that targeting stromal cell processes with cellular therapy has the potential to retard prostate tumor growth.

Fibrillar collagen inhibits cholesterol biosynthesis in human aortic smooth muscle cells.

OBJECTIVE: Integrin-mediated cell adhesion to type I fibrillar collagen regulates gene and protein expression, whereas little is known of its effect on lipid metabolism. In the present study, we examined the effect of type I fibrillar collagen on cholesterol biosynthesis in human aortic smooth muscle cells (SMCs). METHODS AND RESULTS: SMCs were cultured on either fibrillar or monomer collagen for 48 hours and [(14)C]-acetate incorporation into cholesterol was evaluated. Fibrillar collagen reduced by 72.9+/-2.6% cholesterol biosynthesis without affecting cellular cholesterol levels. Fibrillar collagen also reduced 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA) promoter activity (-72.6+/-7.3%), mRNA (-58.7+/-6.4%), protein levels (-35.5+/-8.5%), and enzyme activity (-37.7+/-2.2%). Intracellular levels of the active form of sterol regulatory element binding proteins (SREBP) 1a was decreased by 60.7+/-21.7% in SMCs cultured on fibrillar collagen, whereas SREBP2 was not significantly affected (+12.1+/-7.1%). The overexpression of the active form of SREBP1a rescued the downregulation of fibrillar collagen on HMG-CoA reductase levels. Blocking antibody to alpha2 integrin partially reversed the downregulation of HMG-CoA reductase mRNA expression. Finally, fibrillar collagen led to an intracellular accumulation of unprenylated Ras. CONCLUSIONS: Our study demonstrated that alpha2 beta 1 integrin interaction with fibrillar collagen affected the expression of HMG-CoA reductase, which led to the inhibition of cholesterol biosynthesis in human SMCs.

Targeting stromal-cancer cell interactions with siRNAs.

Tumors are composed of both malignant and normal cells, including fibroblasts, endothelial cells, mesenchymal stem cells, and inflammatory immune cells such as macrophages. These various stromal components interact with cancer cells to promote growth and metastasis. For example, macrophages, attracted by colony-stimulating factor-1 (CSF-1) produced by tumor cells, in turn produce various growth factors such as vascular endothelial growth factor, which supports the growth of tumor cells and their interaction with blood vessels leading to enhanced tumor cell spreading. The activation of autocrine and paracrine oncogenic signaling pathways by stroma-derived growth factors and cytokines has been implicated in promoting tumor cell proliferation and metastasis. Furthermore, matrix metalloproteinases (MMPs) derived from both tumor cells and the stromal compartment are regarded as major players assisting tumor cells during metastasis. Collectively, these recent findings indicate that targeting tumor-stroma interactions is a promising strategy in the search for novel treatment modalities in human cancer. This chapter summarizes our current understanding of the tumor microenvironment and highlights some potential targets for therapeutic intervention with small interfering RNAs.

Colon cancer cell-derived tumor necrosis factor-alpha mediates the tumor growth-promoting response in macrophages by up-regulating the colony-stimulating factor-1 pathway.

The interplay between malignant and stromal cells is essential in tumorigenesis. We have previously shown that colony-stimulating factor (CSF)-1, matrix metalloprotease (MMP)-2, and vascular endothelial growth factor (VEGF)-A production by stromal cells is enhanced by CSF-1-negative SW620 colon cancer cells. In the present study, the mechanisms by which colon cancer cells up-regulate host factors to promote tumorigenesis were investigated. Profiling of tumor cell cytokine expression in SW620 tumor xenografts in nude mice showed increased human tumor necrosis factor (TNF)-alpha mRNA expression with tumor growth. Incubation of macrophages with small interfering (si) RNAs directed against TNF-alpha or TNF-alpha-depleted SW620 cell conditioned medium versus SW620 cell conditioned medium failed to support mouse macrophage proliferation, migration, and expression of CSF-1, VEGF-A, and MMP-2 mRNAs. Consistent with these results, human TNF-alpha gene silencing decreased mouse macrophage TNF-alpha, CSF-1, MMP-2, and VEGF-A mRNA expression in macrophages cocultured with human cancer cells. In addition, inhibition of human TNF-alpha or mouse CSF-1 expression by siRNA reduced tumor growth in SW620 tumor xenografts in mice. These results suggest that colon cancer cell-derived TNF-alpha stimulates TNF-alpha and CSF-1 production by macrophages, and that CSF-1, in turn, induces macrophage VEGF-A and MMP-2 in an autocrine manner. Thus, interrupting tumor cell-macrophage communication by targeting TNF-alpha may provide an alternative therapeutic approach for the treatment of colon cancer.

Colony-stimulating factor-1 transfection of myoblasts improves the repair of failing myocardium following autologous myoblast transplantation.

AIMS: Skeletal myoblasts are used in repair of ischaemic myocardium. However, a large fraction of grafted myoblasts degenerate upon engraftment. Colony-stimulating factor-1 (CSF-1) accelerates myoblast proliferation and angiogenesis. We hypothesized that CSF-1 overexpression improves myoblast survival and cardiac function in ischaemia-induced heart failure. METHODS AND RESULTS: Three weeks following myocardial infarction, rats developed heart failure and received intramyocardial injections of mouse CSF-1-transfected or untransfected primary autologous rat myoblasts, recombinant human CSF-1, mouse CSF-1 expressing plasmids, or culture medium. Tissue gene and protein expression was measured by quantitative RT-PCR (reverse transcription-polymerase chain reaction) and western blotting. Fluorescence imaging and immunocytochemistry were used to analyse myoblasts, endothelial cells, macrophages, and infarct wall thickening. Electrocardiograms were recorded online using a telemetry system. Left ventricular function was assessed by echocardiography over time, and improved significantly only in the CSF-1-overexpressing myoblast group. CSF-1-overexpression enhanced myoblast numbers and was associated with an increased infarct wall thickness, enhanced angiogenesis, increased macrophage recruitment and upregulated matrix metalloproteases (MMP)-2 and -12 in the zone bordering the infarction. Transplantation of CSF-1-overexpressing myoblasts did not result in major arrhythmias. CONCLUSION: Autologous intramyocardial transplantation of CSF-1 overexpressing myoblasts might be a novel strategy in the treatment of ischaemia-induced heart failure.

Modulation of tumor associated macrophages in solid tumors.

It is becoming increasingly clear that interactions between cancer cells, stroma cells and the extracellular matrix (ECM) are pivotal to the processes of neovascularization and tumorigenesis. As tumor stromal components known as tumor associated macrophages (TAMs), mononuclear phagocytes can play a key role in tumor type specific neoangiogenesis by promoting remodeling of the ECM through the production of matrix metalloproteases (MMPs), secreting pro-angiogenic growth factors and stabilizing the tumor vasculature. The growth factor colony-stimulating factor-1 is produced by a wide variety of cancer cells and tumor stroma cells and influences the migration, survival and phenotype of TAMs. Thus, understanding the relationships of the cancer cell with the host environment is key for specifically exploiting tumor growth promoting tumor host interactions for new therapeutic strategies. This review outlines the strategies for targeting CSF-1 in malignancies to influence TAMs in tumor development.

Host CD147 blockade by small interfering RNAs suppresses growth of human colon cancer xenografts.

Tumor cells can stimulate matrix metalloproteinase (MMP) production by stromal cells through cell-cell interactions mediated by cell adhesion molecules such as extracellular matrix metalloproteinase inducer (human CD147/EMMPRIN, mouse CD147/Basigin). This study sought to characterize whether specific tumor-stromal cell interactions mediated by CD147 promote colon cancer growth by utilizing small interfering (si)RNAs directed against human CD147/EMMPRIN or mouse CD147/Basigin in co-cultures of cancer cells with macrophages and fibroblasts and established human SW620 colon cancer xenograft models in immune deficient mice. We show that blockade of host (mouse) CD147/Basigin expression, but not cancer cell-derived CD147/EMMPRIN, suppresses tumor growth in human colon cancer xenografts. Experiments in vitro indicated that colon cancer cell-stromal cell interactions mediated by CD147 lead to increased MMP-2 expression in fibroblasts but not macrophages. Furthermore, expression of host VEGF-A in both fibroblasts and macrophages is independent of CD147 in vitro and in vivo. Interestingly, inhibition of cancer cell-derived EMMPRIN leads to increased MMP-9 levels in vivo. Our findings provide new insights into CD147-mediated tumor-host interactions mediating colon cancer growth.

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 Ringer's 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.

Target validation using RNA interference in solid tumors.

Reverse genetics is one strategy that is currently used to establish a link between a target gene and a disease phenotype. In this process, the function of a gene is inhibited and the consequence of its loss on a desired biological function, such as tumor growth and metastasis, is monitored. RNA interference (RNAi) has been found to be the most effective method to specifically inhibit gene expression. Notably, interactions between cancer cells, stromal cells, and the extracellular matrix (ECM) are crucial to angiogenesis and tumorigenesis. Tumor cells and the surrounding stroma are the principle source of growth factors and cytokines, which induce remodeling of the ECM mediated by metalloproteases (MMPs) secreted by macrophages. The production of macrophages is regulated by colony-stimulating factor (CSF)-1, which is overexpressed in several tumors. When short-interfering RNAs (siRNAs) targeting either the CSF-1 or its receptors were delivered into colon and breast cancer xenografts in mice, tumor growth was inhibited. Associated with this suppression, we observed decreased tumor vascularity, reduced expression of angiogenic factors and MMPs, and decreased macrophage recruitment to the tumors. The suppression of CSF-1 by RNA interference is therefore a powerful tool to block gene function and influence tumor-stroma interactions in solid tumor development.

Vascular endothelial growth factor increases pulmonary vascular permeability in cystic fibrosis patients undergoing lung transplantation.

OBJECTIVE: Vascular endothelial growth factor (VEGF) is the prime regulator of angiogenesis and vascular permeability and its serum levels increase in cystic fibrosis (CF). The mechanisms of VEGF overproduction and its impact on CF lung pathology and pulmonary vascular permeability during lung transplantation are not fully understood. METHODS: The expression of VEGF, its receptors, hypoxia inducible factor (HIF)-1alpha, beta, angiopoietins, and endothelial cell marker CD31 were studied in lung biopsies of CF and COPD patients and controls, using real time reverse transcription (RT)-PCR and Western blotting. DNA binding activity of HIF-1 to VEGF-A promoter was assessed by electrophoretic mobility shift assay (EMSA) and wet-to-dry lung weight ratios as well as microvascular density (MVD) were determined. Serum VEGF-A concentrations in enzyme-linked immunosorbent assay (ELISA) and wet-to-dry weight ratios of donor lungs were monitored during transplantation in CF and COPD patients. Primary graft dysfunction (PGD) was diagnosed and graded according to the guidelines of the International Society for Heart and Lung Transplantation. RESULTS: VEGF-A165 and Flt-1 mRNA expression (P<0.05), VEGF-A (P<0.05), and HIF-1alpha (P<0.05) protein levels, DNA binding activity of HIF-1 to VEGF promoter (P<0.001) and extravascular lung water content (P<0.05) were increased in CF lungs versus controls, whereas MVD was unchanged. Before and during lung transplantation, VEGF-A serum concentrations were higher in CF versus COPD patients (P<0.05) and 60 min following reperfusion donor lungs transplanted to CF patients had higher tissue water contents than in COPD patients (P<0.05). PGD grade 3 occurred more frequently in CF (22.7%) versus COPD patients (4%). PGD grade 3 patients had significantly higher VEGF serum concentrations versus PGD grade 0-2 patients (P<0.001). CONCLUSIONS: These data indicate that upregulated VEGF-A levels are most likely induced by enhanced HIF-1 binding to VEGF-A promoter, possibly contributing to elevated serum VEGF-A levels in CF. Furthermore, CF patients undergoing lung transplantation are possibly more susceptible to PGD because of increased VEGF-A expression that mediates increased lung graft vascular permeability.