Photodynamic therapy with a 5-ALA patch does not increase the risk of conversion of actinic keratoses into squamous cell carcinoma: Results of a multicentre non-interventional study.

BACKGROUND: It is important to collect data about the risk of transformation of an actinic keratosis (AK) lesion into squamous cell carcinoma (SCC) after a single photodynamic therapy (PDT) with 5-ALA patch for a longer follow-up period under daily routine. QUESTIONS ADDRESSED: The purpose of this non-interventional study (NIS) was to collect data on the frequency of occurrence of SCCs in the treated area during an interval of 2 years after a single 5-ALA patch-PDT. EXPERIMENTAL DESIGN: This prospective observational case-only study included patients with mild AK lesions on the head and face treated with 5-ALA patch-PDT according to the Summary of Product Characteristics (SPC). RESULTS: In 370 patients, the risk of transformation of their treated AK lesion into SCC was 0.073% with its exact 95% confidence interval using the Poisson distribution of [0.009%, 0.262%]. The rate of complete clinical clearance on lesion basis after 3 months was 84.3%. CONCLUSION: The efficacy and the safety results show no observation of an increased risk for conversion of an AK into a SCC 2 years after a single 5-ALA patch-PDT. Additionally, the high clinical complete remission rate under routine conditions is comparable to the rates observed in the approval trials.

AP1-dependent repression of TGFα-mediated MMP9 upregulation by PPARδ agonists in keratinocytes.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that function mainly in the regulation of glucose and lipid homeostasis. PPAR agonists have been shown to control inflammation by inhibition of distinct proinflammatory genes. Aberrant activation of the epidermal growth factor receptor and/or overexpression of its ligand, transforming growth factor-α (TGFα), are key features of both neoplastic and inflammatory hyperproliferative epithelia. Matrix metalloproteinase 9 (MMP9) belongs to the set of genes that are effectively induced by TGFα in keratinocytes. Induction of MMP9 expression is strongly linked to regenerative skin repair mechanisms, inflammatory skin diseases and tumor metastasis. We explored whether the known anti-inflammatory effects of PPARδ ligands involve inhibiting the TGFα-mediated upregulation of MMP9. The PPARδ agonists potently inhibited TGFα-induced MMP9 expression in human keratinocytes. This inhibition was observed at both the protein and mRNA levels. Transcriptional activation studies with deletion constructs of a reporter gene revealed that PPARδ agonists mediate their inhibitory effects via an AP1-binding site. Electromobility shift assay analysis indicated that MMP9 gene expression is inhibited by repressing site-dependent DNA binding and transactivation by c-fos. In conclusion, our data provide the first evidence that MMP9 expression induced by TGFα is a valid target of PPARδ ligands in keratinocytes.

Suppression of VEGFR2 expression in human endothelial cells by dimethylfumarate treatment: evidence for anti-angiogenic action.

The association between angiogenesis and chronic inflammatory diseases, such as psoriasis, seems to be an important phenomenon implicated in the pathogenesis of these medical conditions. Recent studies provide evidence that dimethylfumarate (DMF) has a profound anti-inflammatory as well as anti-tumorigenic action, although the effect of DMF on angiogenesis is unknown. Signaling via the vascular endothelial growth factor receptor-2 (VEGFR2) pathway is critical for angiogenic responses. Therefore, we explored whether the known anti-inflammatory and anti-tumorigenic properties of DMF might be mediated in part by anti-angiogenic effects through the reduction in VEGFR2 expression. In this study, DMF was found to inhibit endothelial VEGFR2 expression; time- and concentration-dependent inhibition was demonstrated both at the level of protein and mRNA expression. This blockade was coincident with the inhibition of the formation of capillary-like structures. The DMF-dependent inhibition of VEGFR2 transcription was found to be mediated by an element located between base pairs -60 and -37, which contains two adjacent, consensus Sp1 transcription factor-binding sites, and the constitutive formation of complexes containing Sp1 at this site is decreased by DMF treatment. Inhibition of VEGFR-2 is shown to be one critical aspect in DMF-mediated anti-angiogenic effects.

Peroxisome proliferator-activated receptor {delta} activators induce IL-8 expression in nonstimulated endothelial cells in a transcriptional and posttranscriptional manner.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that are implicated in the regulation of lipid and glucose homeostasis. PPAR agonists have been shown to control inflammatory processes, in part by inhibiting distinct proinflammatory genes (e.g. Il-1ß and IFN-γ). IL-8 is a member of the proinflammatory chemokine family that is important for various functions, such as mediating the adhesion of eosinophilic granulocytes onto endothelial cells. The influence of PPARδ activators on the expression of IL-8 in noninduced quiescent endothelial cells is unclear. Therefore, we explored the influence of PPARδ activators on the expression of IL-8 in nonstimulated endothelial cells. PPARδ agonists induce IL-8 expression in human umbilical vein endothelial cells. This induction is demonstrated at the level of both protein and mRNA expression. Transcriptional activation studies using IL-8 reporter gene constructs and DNA binding assays revealed that PPARδ agonists mediated their effects via an NFκB binding site. It is well known that IL-8 is also regulated by mRNA stability. To provide further evidence for this concept, we performed mRNA stability assays and found that PPARδ agonists induce the mRNA stability of IL-8. In addition, we showed that PPARδ agonists induce the phosphorylation of ERK1/2 and p38, which are known to be involved in the increase of mRNA stability. The inhibition of these MAPK signaling pathways resulted in a significant suppression of the induced IL-8 expression and the reduced mRNA stability. Therefore, our data provide the first evidence that PPARδ induces IL-8 expression in nonstimulated endothelial cells via transcriptional as well as posttranscriptional mechanisms.

Bone marrow derived cells in the tumour microenvironment contain cells with primitive haematopoietic phenotype.

Infiltration of bone marrow derived cells is part of the angiogenic switch required for uncontrolled tumour growth. However, the nature of the tumour-infiltrating cells from bone marrow has not been fully elucidated. To investigate the phenotype of bone marrow derived cells within a tumour, we employed the Lewis lung carcinoma (LLC) murine tumour model. We followed bone marrow derivation of tumour-infiltrating cells through transplantation of CD45.2 bone marrow cells into pre-irradiated CD45.1 mice. We found robust CD45.2 donor type chimerism in bone marrow and blood of CD45.1 recipient tumour-bearing mice. Flow cytometric analysis of LLC tumours showed, in addition to previously described pro-angiogenic CD45(+)VEGFR2(+)'endothelial progenitor cells' (EPC), or CD45(+)Tie2(+)'Tie2-expressing monocytes' (TEM), incorporation of donor type lineage marker negative (Lin(-)) and Lin(-)Sca1(+) undifferentiated haematopoietic cell types. Immunohistochemical analysis confirmed the extravasal location of the primitive haematopoietic cells. Flow-cytometric sorting of bone marrow cells and subsequent analysis in haematopoietic colony-forming assays revealed that cells with a Lin(-)Sca1(+) phenotype, which were initially negative for VEGFR2 and Tie2, gave rise to VEGFR2(+) and/or Tie2(+) cells. Moreover, Lin(-) bone marrow cells pre-labelled with the membrane dye PKH26 (a red fluorochrome) and transplanted i.v. into tumour-bearing mice were found to extravasate and incorporate into LLC tumours within 24 hrs. Thus, primitive haematopoietic precursors which are thought to be precursors of EPC and TEMs, constitute a part of the tumour microenvironment. This makes them an attractive target cell population for tumour-directed cellular therapies.

Inhibition of Rac1 GTPase downregulates vascular endothelial growth factor receptor-2 expression by suppressing Sp1-dependent DNA binding in human endothelial cells.

RhoA, Rac1 and CDC42 are small GTP-binding proteins of the Rho family that play a crucial role in regulation of the actin-based cytoskeleton. In addition to cell growth regulation, they are implicated in transcriptional activation, oncogenic transformation and angiogenesis. The small Rho-GTPases have been linked to vascular endothelial growth factor (VEGF)-induced signalling pathways, but their role has not yet been elucidated. As signalling via the VEGF receptor-2 (VEGFR2) pathway is critical for angiogenic responses in cancer, wound repair and ischaemic and inflammatory diseases, we investigated whether the small Rho-GTPase Rac1 influences VEGFR2 expression in human endothelial cells. In this study, we show that a dominant negative Rac1 expression vector led to a pronounced decrease in VEGFR2 mRNA and protein expression. To identify minimal promoter requirements and potential applications of the small Rho-GTPases, we used VEGFR2 promoter-reporter gene constructs containing various deletions. The inhibitory effects of dominant negative Rac1 on the transcriptional activity of the VEGFR2 promoter localized to an element between -77 and -60 that contains an Sp1 transcription factor binding site. Electrophoretic mobility shift assays demonstrated that constitutive Sp1-dependent DNA binding decreased with Rac1 inhibition. Hence, repression of the small Rho GTPase Rac1 seems to be an additional critical molecular mechanism in the regulation of VEGFR2 expression.

Results of a phase III, randomized, placebo-controlled study of sorafenib in combination with carboplatin and paclitaxel as second-line treatment in patients with unresectable stage III or stage IV melanoma.

PURPOSE: This phase III, randomized, double-blind, placebo-controlled study was conducted to evaluate the efficacy and safety of sorafenib with carboplatin and paclitaxel (CP) in patients with advanced melanoma who had progressed on a dacarbazine- or temozolomide-containing regimen. PATIENTS AND METHODS: A total of 270 patients were randomly assigned to receive intravenous paclitaxel 225 mg/m2 plus intravenous carboplatin at area under curve 6 (AUC 6) on day 1 of a 21-day cycle followed by either placebo (n = 135) or oral sorafenib 400 mg (n = 135) twice daily on days 2 to 19. The primary efficacy end point was progression-free survival (PFS); secondary and tertiary end points included overall survival and incidence of best response, respectively. RESULTS: The median PFS was 17.9 weeks for the placebo plus CP arm and 17.4 weeks for the sorafenib plus CP arm (hazard ratio, 0.91; 99% CI, 0.63 to 1.31; two-sided log-rank test P = .49). Response rate was 11% with placebo versus 12% with sorafenib. Dermatologic events, grade 3 thrombocytopenia, diarrhea, and fatigue were more common in patients treated with sorafenib plus CP versus placebo plus CP. CONCLUSION: In this study, the addition of sorafenib to CP did not improve any of the end points over placebo plus CP and cannot be recommended in the second-line setting for patients with advanced melanoma. Both regimens had clinically acceptable toxicity profiles with no unexpected adverse events. A trial of similar design for the first-line treatment of patients with advanced melanoma (intergroup trial E2603) is currently ongoing.

Down-regulation of vascular endothelial growth factor receptor 2 is a major molecular determinant of proteasome inhibitor-mediated antiangiogenic action in endothelial cells.

The ubiquitin-proteasome system is the major pathway for intracellular protein degradation in eukaryotic cells. This system controls a wide range of cellular regulatory proteins, including transcription factors and cell cycle regulatory proteins. Recent evidence also established the importance of the proteasome in tumor development, showing antitumor and antiangiogenic actions by using selective inhibitors in vivo. As signaling via the vascular endothelial growth factor receptor 2 (VEGFR2) pathway is critical for angiogenic responses to occur, we explored whether antiangiogenic effects due to proteasome inhibition were partly mediated through decreased endothelial VEGFR2 expression. This study shows that different proteasome inhibitors blocked VEGFR2 expression in a time-dependent and concentration-dependent manner. This blockade was paralleled by the respective inhibition of the formation of capillary-like structures and endothelial cell migration. In contrast, neither tie-2 nor VEGFR1 expression was significantly affected by proteasome inhibitor treatment. The suppressive effects on VEGFR2 expression were not conveyed by increased shedding or a decrease in protein half-life, suggesting that transcriptional mechanisms accounted for the observed effects. In line with this conclusion, proteasome inhibition significantly suppressed VEGFR2 mRNA accumulation. In addition, inhibitor treatment considerably decreased the transcriptional activity of 5' deletional VEGFR2 promoter gene constructs. Proteasome inhibition-mediated repression was controlled by a GC-rich region that harbored one consensus Sp1-binding site. Subsequent EMSA analyses showed decreased constitutive Sp1-dependent DNA binding in response to proteasome inhibition. In addition, we could show that proteasome inhibitors reduced VEGFR2 mRNA stability. Therefore, VEGFR2 expression may constitute a critical molecular target of proteasome inhibitors that may mediate their antiangiogenic effects in vivo.

Microtubule-targeted drugs inhibit VEGF receptor-2 expression by both transcriptional and post-transcriptional mechanisms.

Cytoskeletal polymers control a wide range of cellular functions, including proliferation, migration, and gene expression. As changes in endothelial cell shape and motility are required to form vascular networks, we hypothesized that disassembly of actin filaments or microtubules may impact endothelial vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2) expression as a critical determinant of angiogenesis. We therefore investigated the effect of actin filament- and microtubule-disrupting agents on VEGFR1 and VEGFR2 expression by endothelial cells. Microtubule (MT) disassembly greatly inhibited endothelial VEGFR2 expression, whereas VEGFR1 expression levels remained largely unchanged. These suppressive effects were neither conveyed by increased VEGFR2 shedding nor by shortened protein half-life, suggesting that transcriptional mechanisms account for the observed effects. In line with this conclusion, MT disruption significantly suppressed endothelial VEGFR2 mRNA accumulation. The treatment considerably decreased transcriptional activity of 5'-deletional VEGFR2 promoter gene constructs. MT disruption-mediated repression was conveyed by a GC-rich region harboring two consensus Sp1-binding sites. Electrophoretic mobility-shift assay analysis demonstrated that constitutive Sp1-dependent DNA binding is decreased by MT disassembly. In addition, we provide evidence for additional post-transcriptional regulatory mechanisms, as the VEGFR2 mRNA half-life is significantly reduced by MT-disrupting agents. Hence, both inhibition of the rate of gene transcription and increased mRNA turnover represent critical molecular mechanisms by which MT disruption inhibits VEGFR2 expression.

Simultaneous blockade of VEGFR-1 and VEGFR-2 activation is necessary to efficiently inhibit experimental melanoma growth and metastasis formation.

Metastasis continues to be the major cause of morbidity and mortality in malignant melanoma. In our study, we explored whether inhibition of VEGFR-1 or VEGFR-2 signaling conveys distinct suppressive effects on B16 melanoma subcutaneous growth and metastasis formation. The inhibition of VEGFR-1 or -2 alone had no significant influence on both melanoma growth and metastasis formation. In contrast, simultaneous blockade of VEGFR-1 and -2 signaling strongly suppressed progression in both B16 tumor models. There was no expression of VEGFR-1 or -2 detectable on the B16 cells used, excluding the melanoma cells as direct therapeutic targets. Analyzing the contribution of progenitor-like cells during melanoma metastasis formation, we observed an enhanced proliferation and mobilization of VEGFR-1+ myeloid and VEGFR-2+ endothelial cells with progenitor potential by the induction of melanoma lung metastasis, which was not influenced by interference with VEGFR signaling. These results indicate that the antimetastatic effects exerted by combined inhibition of VEGFR-1 and -2 signaling were mediated via targeting cell populations other than progenitors only. Sole inhibition of VEGFR-1 signaling led to a strong reduction of the CD45-positive inflammatory infiltrate in the tumor tissue. However, the formation of lung metastasis was not affected, indicating that inhibition of the inflammatory response was not sufficient to efficiently block B16 melanoma metastasis development. Taken together, our data suggest that in the utilized B16 tumor models the blockade of both the inflammatory and the VEGFR-2-dependent angiogenic response are necessary to effectively inhibit solid tumor growth and formation of lung metastasis by B16 melanoma cells.

Mesenchymal stem cells display coordinated rolling and adhesion behavior on endothelial cells.

To explore the initial steps by which transplanted mesenchymal stem cells (MSCs) interact with the vessel wall in the course of extravasation, we studied binding of human MSCs to endothelial cells (ECs). In a parallel plate flow chamber, MSCs bound to human umbilical vein ECs (HUVECs) similar to peripheral-blood mononuclear cells (PBMCs) or CD34(+) hematopoietic progenitors at shear stresses of up to 2 dynes/cm(2). This involved rapid extension of podia, rolling, and subsequent firm adhesion that was increased when ECs were prestimulated with TNF-alpha. MSC binding was suppressed when ECs were pretreated with function-blocking anti-P-selectin antibody, and rolling of MSCs was induced on immobilized P-selectin, indicating that P-selectin was involved in this process. Preincubation of HUVECs with anti-VCAM-1 or of MSCs with anti-VLA-4 antibodies suppressed binding of MSCs to HUVECs but did not enhance inhibition by anti-P-selectin, indicating that both P-selectin and VCAM-1 are equally required for this process. Intravital microscopy demonstrated the capacity of MSCs to roll and adhere to postcapillary venules in vivo in a mouse model in a P-selectin-dependent manner. Thus, MSCs interact in a coordinated fashion with ECs under shear flow, engaging P-selectin and VCAM-1/VLA-4.

[Dystrophic calcinosis cutis after subcutaneous administration of para-aminosalicylic acid for treatment of pulmonary tuberculosis]. / Dystrophe Calcinosis cutis nach subkutaner Verabreichung von Para-Amino-Salizylsäure zur Behandlung einer Lungentuberkulose.

An 81-year-old women presented with monstrous subcutaneous mobile masses on the ventral and ventrolateral surfaces of her thighs. Radiography as well as computer tomography showed extensive soft tissue calcifications. We interpreted these alterations as dystrophic calcinosis cutis, most likely resulting from repeated subcutaneous PAS infusions for the treatment of pulmonary tuberculosis.

The ability of different forms of heparins to suppress P-selectin function in vitro correlates to their inhibitory capacity on bloodborne metastasis in vivo.

Ample evidence suggests that many of the in vivo anti-metastatic effects by heparins reflect their actions on P-selectin-mediated binding. We hypothesized that the ability of widely used heparins and derivatives to interfere with P-selectin-dependent tumour cell interactions under flow in vitro could be used to identify anticoagulants with advanced inhibitory functions on experimental blood-borne metastasis in vivo. To test this assumption, the impact of unfractionated heparin, the low-molecular-weight heparins (LMWH) nadroparin and enoxaparin, and the synthetic pentasaccharide fondaparinux on P-selectin-dependent tumour interactions in vitro and metastasis formation in vivo were evaluated. Our data revealed that these commonly used anticoagulants widely differ in their potential to interfere with P-selectinmediated cell binding. Importantly, the superior inhibitory capacity on P-selectin function of unfractionated heparin and LMWH nadroparin as opposed to LMWH enoxaparin and synthetic heparin pentasaccharide fondaparinux strongly correlated to the inhibitory potency of each in inhibiting experimental lung metastasis in vivo. Hence, P-selectin inhibition may constitute a valuable feature to identify anticoagulants that are suitable for anticancer therapy.

Antiangiogenic cancer therapies get their act together: current developments and future prospects of growth factor- and growth factor receptor-targeted approaches.

Targeting the vascular endothelial growth factor (VEGF) in combination with standard chemotherapy has recently proved successful in the treatment of different types of advanced cancer. The achievements of combinatorial anti-VEGF monoclonal antibody bevacizumab (BEV) renewed the confidence in targeted antiangiogenic approaches to constitute a complementary therapeutic modality in addition to surgery, radiotherapy and chemotherapy. While several second-generation multitargeted tyrosine kinase inhibitors show promise in defined tumor entities, these novel antiangiogenic compounds have yet to meet or exceed the efficacy of combinatorial BEV therapy in ongoing clinical trials. Current developments of targeted antiangiogenic agents include their use in the adjuvant setting and the combination of different antiangiogenesis inhibitors to take a more comprehensive approach in blocking tumor angiogenesis. The identification of surrogate markers that can monitor the activity and efficacy of antiangiogenic drugs in patients belongs to the most critical challenges to exploit the full potential of antiangiogenic therapies. The opportunities and obstacles in further development of growth factor- and growth factor receptor-targeted antiangiogenic approaches for advanced cancer, including malignant melanoma, will be discussed herein with particular reference to selected ongoing clinical trials.

Role of the monomeric GTPase Rho in hematopoietic progenitor cell migration and transplantation.

To investigate the role of the monomeric guanosine triphosphatase (GTPase) Rho on migration of hematopoietic progenitor cells (HPC), we employed different clostridial toxins which inhibit the Rho family of GTPases. Pretreatment with C2I-C3, a cell-accessible C3 transferase fusion protein that targets Rho, increased chemokinetic migration of the factor-dependent multipotent cell line Factor Dependent Cell Paterson with mixed lineage differentiation potential (FDCP-mix) and of primary lineage marker-depleted HPC in vitro. In contrast, treatment with lethal toxin (LT) from Clostridium sordellii, which predominantly inactivates Rac, and with toxin B from C. difficile, which inactivates Rho, Rac and Cdc42, decreased in vitro migration. When HPC pretreated with LT or toxin B were transplanted into mice, homing to the bone marrow was impaired, whereas C2I-C3 treatment did not alter HPC homing. However, in a competitive hematopoietic repopulation experiment in C57BL/6 mice, pretreatment of bone marrow cells with any of the inhibitors, including the Rho inhibitor C2I-C3, resulted in suppressed donor-type hematopoiesis. Our data indicate that whereas Rac supports HPC cell cycling, migration, short-term homing and hematopoietic regeneration, Rho coordinates down-regulation of HPC migration and is required for hematopoietic regeneration.

Metronomic low-dose chemotherapy as antiangiogenic therapeutic strategy for cancer.

New blood vessel formation is essential for the growth and metastasis of many cancers. As a result, antitumor activities of various angiogenesis inhibitors have been intensely explored in various tumors. Recent preclinical studies suggest that certain conventional cytotoxic agents can function as antiangiogenic drugs when administered at comparatively low doses on a continuous or very frequent schedule. Such antiangiogenic 'metronomic' scheduling of chemotherapy without extended rest periods has been shown to exert significant therapeutic antitumor efficacy with very limited toxicity in different tumor models. Combining metronomic low-dose chemotherapy regimens with specific angiogenesis inhibitors further increases efficacy. Based on the promising preclinical studies, it is anticipated that metronomic chemotherapy in combination with angiogenesis inhibitors will prove effective in clinical trials in terms of survival prolongation. While considerable progress may derive from larger randomized clinical studies, only joint efforts between basic and clinical research will ultimately advance the new paradigm of long-term metronomic antiangiogenic chemotherapy, which carries the prospect of turning cancer into a more controllable chronic disease at minimal toxicity.

Endothelial P-selectin as a target of heparin action in experimental melanoma lung metastasis.

Spontaneous and experimental metastasis can be effectively inhibited by the widely used anticoagulant heparin in different tumor models. At the cellular level, many of the antimetastatic effects of heparin in vivo are due to its action on P-selectin-mediated binding. Whereas previous attention has focused on P-selectin-dependent tumor-cell-platelet interactions in blood-borne metastasis, we sought to address the potential contribution of endothelial P-selectin expression to adhesive events between the microvasculature and melanoma cells in vivo. Transplantation of bone marrow from P-selectin-deficient into wild-type mice conveyed inhibition of ex-perimental melanoma metastasis. However, the extent to which bone marrow-conferred lack of platelet P-selectin expression attenuated melanoma lung metastasis was significantly less than that seen in P-selectin-deficient mice, suggesting that endothelial P-selectin expression may additionally contribute to formation of hematogenous metastases. This assumption was supported by our intravital microscopy studies, in which a significant proportion of melanoma cells were capable of directly interacting with postcapillary venules of the murine ear in a P-selectin-dependent manner. Heparin not only inhibits P-selectin-mediated melanoma cell rolling but also attenuates melanoma metastasis formation in vivo, further supporting the concept that endothelial P-selectin expression may represent an additional target of heparin action in experimental melanoma lung metastasis.

PPARalpha activators inhibit vascular endothelial growth factor receptor-2 expression by repressing Sp1-dependent DNA binding and transactivation.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors, originally implicated in the regulation of lipid and glucose homeostasis. In addition, natural and synthetic PPAR activators may control inflammatory processes by inhibition of distinct proinflammatory genes. As signaling via the vascular endothelial growth factor receptor-2 (VEGFR2) pathway is critical for angiogenic responses during chronic inflammation, we explored whether known antiinflammatory effects of PPAR ligands are mediated in part through diminished VEGFR2 expression. In this study, PPARalpha agonists are found to inhibit endothelial VEGFR2 expression, whereas predominant PPARgamma ligands remained without discernible effects. Time- and concentration-dependent inhibition is demonstrated both at the level of protein and mRNA VEGFR2 expression. Inhibitory effects of PPARalpha agonists on transcriptional activity of the VEGFR2 promoter are conveyed by an element located between base pairs -60 and -37 that contains two adjacent consensus Sp1 transcription factor binding sites. Constitutive Sp1-containing complex formation to this sequence is decreased by PPARalpha treatment, indicating that VEGFR2 gene expression is inhibited by repressing Sp1 site-dependent DNA binding and transactivation. Our coimmunoprecipitation experiments revealed enhanced protein interactions between PPARalpha and Sp1 on PPARalpha activation, thus constituting a probable mechanism by which PPARalpha activators decrease Sp-dependent binding activity to the VEGFR2 promoter. Hence, molecular mechanisms by which PPARs modulate the rate of gene transcription may include direct interactions between specific transcription factors and PPARs that ultimately result in reduced DNA binding to their respective response elements.