Secretion Of Angiogenic Growth Factors Research Articles

Fish oil-enriched diet protects against ischemia by improving angiogenesis, endothelial progenitor cell function and postnatal neovascularization

BackgroundFish oil consumption has been associated with a reduced incidence of cardiovascular diseases. However, the precise mechanisms involved are not completely understood. Here we tested the hypothesis that a fish oil-enriched diet improves neovascularization in response to ischemia. Methods and resultsC57Bl/6 mice were fed a diet containing either 20% fish oil, rich in long-chain n-3 polyunsaturated fatty acids (PUFAs), or 20% corn oil, rich in n-6 PUFAs. After 4 weeks, hindlimb ischemia was surgically induced by femoral artery removal. We found that blood flow recovery was significantly improved in mice fed a fish oil diet compared to those fed a corn oil diet (Doppler flow ratio (DFR) at day 21 after surgery 78 ± 5 vs. 56 ± 4; p < 0.01). Clinically, this was associated with a significant reduction of ambulatory impairment and ischemic damage in the fish oil group. At the microvascular level, capillary density was significantly improved in ischemic muscles of mice fed a fish oil diet. This correlated with increased expression of VEGF and eNOS in ischemic muscles, and higher NO concentration in the plasma. Endothelial progenitor cells (EPCs) have been shown to have an important role for postnatal neovascularization. We found that the number of EPCs was significantly increased in mice fed a fish oil diet. In addition, oxidative stress levels (DCF-DA, DHE) were reduced in EPCs isolated from mice exposed to fish oil, and this was associated with improved EPC functional activities (migration and integration into tubules). In vitro, treatment of EPCs with fish oil resulted in a significant increase of cellular migration. In addition, the secretion of angiogenic growth factors including IL6 and leptin was significantly increased in EPCs exposed to fish oil. ConclusionFish oil-enriched diet is associated with improved neovascularization in response to ischemia. Potential mechanisms involved include activation of VEGF/NO pathway in ischemic tissues together with an increase in the number and the functional activities of EPCs.

PM.20 Thyroid Hormone Action in the Decidua During Human Pregnancy

IntroductionMaternal thyroid dysfunction is associated with complications of malplacentation including miscarriages and pre-eclampsia. We hypothesise that thyroid hormones (TH) play an important role within human decidua in regulating placentation.MethodsDeciduas from...

Platelet-Rich Plasma Enhances the Dermal Regeneration Efficacy of Human Adipose-Derived Stromal Cells Administered to Skin Wounds

The administration of human adipose-derived stromal cells (hASCs) enhances skin wound healing. However, poor survival of hASCs that are administered to avascular wound regions may limit the therapeutic efficacy of the hASCs. The aim of this study was to determine whether the coadministration of platelet-rich plasma (PRP) and hASCs enhanced the skin wound-healing efficacy of hASCs. Skin regeneration was examined in skin wounds of athymic mice that were either untreated or treated with hASCs, PRP, or both hASCs and PRP. Coadministration of PRP and hASCs resulted in better skin regeneration than hASC administration alone in part by significantly improving the proliferation of administered hASCs by the angiogenic growth factor secretion of the hASCs and surrounding mouse host cells in the wound areas and by promoting neovascularization in the wound beds.

Functionalized self-assembling peptide nanofiber hydrogels mimic stem cell niche to control human adipose stem cell behavior in vitro

A class of designer functionalized self-assembling peptide nanofiber scaffolds developed from self-assembling peptide RADA16-I (AcN-RADARADARADARADA-CONH2) has become increasingly attractive not only for studying spatial behaviors of cells, but also for developing approaches for a wide range of medical applications including regenerative medicine, rapid hemostasis and cell therapy. In this study, we report three functionalized self-assembling peptide hydrogels that serve as a three-dimensional (3-D) artificial microenvironment to control human adipose stem cell (hASC) behavior in vitro. Short peptide motifs SKPPGTSS (bone marrow homing motif), FHRRIKA (heparin-binding motif) and PRGDSGYRGDS (two-unit RGD cell adhesion motif) were used to extend the C-terminus of RADA16-I to obtain functionalized peptides. Atomic force microscopy confirmed the formation of self-assembling nanofibers in the mixture of RADA16-I peptide and functionalized peptides. The behaviors of hASCs cultured in 3-D peptide hydrogels, including migration, proliferation and growth factor-secretion ability, were studied. Our results showed that the functionalized peptide hydrogels were suitable 3-D scaffolds for hASC growth with higher cell proliferation, migration and the secretion of angiogenic growth factors compared with tissue culture plates and pure RADA16-I scaffolds. The present study suggests that these functionalized designer peptide hydrogels not only have promising applications for diverse tissue engineering and regenerative medicine applications as stem cell delivery vehicles, but also could be a biomimetic 3-D system to study nanobiomaterial–stem cell interactions and to direct stem cell behaviors.

Acceleration of segmental bone regeneration in a rabbit model by strontium-doped calcium polyphosphate scaffold through stimulating VEGF and bFGF secretion from osteoblasts

The development of suitable bioactive three-dimensional scaffold for the promotion of bone regeneration is critical in bone tissue engineering. The purpose of this study was to investigate in vivo osteogenesis of the porous strontium-doped calcium polyphosphate (SCPP) scaffolds for bone repair, as well as the relationship between osteogenic properties of SCPP scaffolds and the secretion of bFGF and VEGF from osteoblasts stimulated by SCPP. Besides, the advantages of scaffolds seeded with mesenchymal stem cells (MSCs) for bone repair were also studied. Firstly, the bone repair evaluation of scaffolds was performed on a rabbit segmental bony defects model over a period of 16weeks by histology combined with X-ray microradiography. And then, in order to avoid the influence from the other factors such as hypoxia which emerge in vivo study and affect the secretion of VEGF and bFGF from host cells, human osteoblast-like cells (MG63) were seeded to SCPP, CPP and HA scaffolds in vitro to determine the ability of these scaffolds to stimulate the secretion of angiogenic growth factors (VEGF and bFGF) from MG63 and further explore the reason for the better osteogenic properties of SCPP scaffolds. The histological and X-ray microradiographic results showed that the SCPP scaffolds presented better osteogenic potential than CPP and HA scaffolds, when combined with MSCs, the SCPP scaffolds could further accelerate the bone repair. And the amounts of VEGF measured by ELISA assay in SCPP, CPP and HA groups after cultured for 7days were about 364.989pg/mL, 244.035pg/mL and 232.785pg/mL, respectively. Accordingly, the amounts of bFGF were about 27.085pg/mL, 15.727pg/mL and 8.326pg/mL. The results revealed that the SCPP scaffolds significantly enhanced the bFGF and VEGF secretion compared with other scaffolds. The results presented in vivo and in vitro study demonstrated that the SCPP could accelerate bone formation through stimulating the secretion of VEGF and bFGF from osteoblasts, making it attractive for bone regeneration.

Locally Administered Adipose-Derived Stem Cells Accelerate Wound Healing through Differentiation and Vasculogenesis

Despite advances in wound closure techniques and devices, there is still a critical need for new methods of enhancing the healing process to achieve optimal outcomes. Recently, stem cell therapy has emerged as a new approach to accelerate wound healing. Adipose-derived stem cells (ASCs) hold great promise for wound healing, because they are multipotential stem cells capable of differentiation into various cell lineages and secretion of angiogenic growth factors. The aim of this study was to evaluate the benefit of ASCs on wound healing and then investigate the probable mechanisms. ASCs characterized by flow cytometry were successfully isolated and cultured. An excisional wound healing model in rat was used to determine the effects of locally administered ASCs. The gross and histological results showed that ASCs significantly accelerated wound closure in normal and diabetic rat, including increased epithelialization and granulation tissue deposition. Furthermore, we applied GFP-labeled ASCs on wounds to determine whether ASCs could differentiate along multiple lineages of tissue regeneration in the specific microenvironment. Immunofluorescent analysis indicated that GFP-expressing ASCs were costained with pan-cytokeratin and CD31, respectively, indicating spontaneous site-specific differentiation into epithelial and endothelial lineages. These data suggest that ASCs not only contribute to cutaneous regeneration, but also participate in new vessels formation. Moreover, ASCs were found to secret angiogenic cytokines in vitro and in vivo, including VEGF, HGF, and FGF2, which increase neovascularization and enhance wound healing in injured tissues. In conclusion, our results demonstrate that ASC therapy could accelerate wound healing through differentiation and vasculogenesis and might represent a novel therapeutic approach in cutaneous wounds.

In vitro study in stimulating the secretion of angiogenic growth factors of strontium-doped calcium polyphosphate for bone tissue engineering

Angiogenesis of tissue-engineered bone remains a limited factor for the engineering of larger bone tissue constructs. Attempts to stimulate angiogenesis, using recombinant protein or gene transfer of angiogenic growth factors, have been proposed; however, these approaches have been associated with some problems regarding such as complex technique, expensive prices as well as safety problems and short half-life of angiogenic growth factors. This study was performed to determine the ability of strontium-doped calcium polyphosphate (SCPP) to induce angiogenesis via researching its effect on the mRNA expressions and protein secretion of VEGF and bFGF in/from cultured osteoblasts (ROS17/2.8 cells). We cultured osteoblasts with SCPP scaffolds containing various doses of strontium as well as calcium polyphosphate (CPP) scaffold. Through the detection of MTT and SEM, we have found that SCPP could promote cell proliferation and maintain their morphology. The results of RT-PCR and ELISA indicated that, compared with those in CPP group, the mRNA expression as well as protein levels of VEGF and bFGF in/from cultured osteoblasts were dose-dependent increasing in response to increasing strontium before reaching the peak in SCPP groups, and 8% SCPP showed the optimal promoting role. Therefore, SCPP containing proper dose of strontium could be served as a potential biomaterial with stimulating angiogenesis in bone tissue engineering and bone repair.

Combined Gene Therapy with Hypoxia-Inducible Factor-1α and Heme Oxygenase-1 for Therapeutic Angiogenesis

Transfection with either hypoxia-inducible factor-1α (HIF-1α) or heme oxygenase-1 (HO-1) gene can induce neovascularization in ischemic tissues. Although expression of transfected HIF-1α gene occurs rapidly, the expressed HIF-1α protein degrades quickly, limiting its therapeutic efficacy. Meanwhile, expressed HO-1 protein does not rapidly undergo degradation, but gene expression occurs a couple of days after transfection, resulting in apoptosis and a delay in angiogenesis in ischemic tissues at the incipient period of HO-1 gene transfection. We hypothesize that combined delivery of HIF-1α and HO-1 gene will enhance antiapoptosis and neovascularization in ischemic tissue compared with HIF-1α or HO-1 single-gene therapy. To test this hypothesis, ischemic mouse hindlimbs were treated with HIF-1α and/or HO-1 gene therapy. The combined gene therapy proved superior to both single-gene therapies, resulting in rapid expression of HIF-1α gene and long-term maintenance of expressed HO-1 protein. The apoptosis in the ischemic region was significantly less, and angiogenic growth factor secretion and angiogenesis were greater in the combined gene therapy than in either of the single-gene therapies. Our results suggest that a combined gene therapy of HIF-1α and HO-1 enhances the transfection of both genes and improves angiogenesis compared with either single-gene therapy.

Regulation of angiogenesis during osseointegration by titanium surface microstructure and energy

Rough titanium (Ti) surface microarchitecture and high surface energy have been shown to increase osteoblast differentiation, and this response occurs through signaling via the α2β1 integrin. However, clinical success of implanted materials is dependent not only upon osseointegration but also on neovascularization in the peri-implant bone. Here we tested the hypothesis that Ti surface microtopography and energy interact via α2β1 signaling to regulate the expression of angiogenic growth factors. Primary human osteoblasts (HOB), MG63 cells and MG63 cells silenced for α2 integrin were cultured on Ti disks with different surface microtopographies and energies. Secreted levels of vascular endothelial growth factor-A (VEGF-A), basic fibroblast growth factor (FGF-2), epidermal growth factor (EGF), and angiopoietin-1 (Ang-1) were measured. VEGF-A increased 170% and 250% in MG63 cultures, and 178% and 435% in HOB cultures on SLA and modSLA substrates, respectively. In MG63 cultures, FGF-2 levels increased 20 and 40-fold while EGF increased 4 and 6-fold on SLA and modSLA surfaces. These factors were undetectable in HOB cultures. Ang-1 levels were unchanged on all surfaces.Media from modSLA MG63 cultures induced more rapid differentiation of endothelial cells and this effect was inhibited by anti-VEGF-A antibodies. Treatment of MG63 cells with 1α,25(OH)2D3 enhanced levels of VEGF-A on SLA and modSLA.Silencing the α2 integrin subunit increased VEGF-A levels and decreased FGF-2 levels. These results show that Ti surface microtopography and energy modulate secretion of angiogenic growth factors by osteoblasts and that this regulation is mediated at least partially via α2β1 integrin signaling.

Secretion of Angiogenic Growth Factors by Villous Cytotrophoblast and Extravillous Trophoblast in Early Human Pregnancy

Angiogenesis is a key feature of placental and uterine development in early pregnancy. We hypothesized that trophoblast cells produce angiogenic growth factors, and that expression differs between villous cytotrophoblast (CTB) and extravillous trophoblast (EVT) cells. Fourteen angiogenic growth factors were measured by multiplex growth factor analysis or ELISA in tissue culture supernatants from EVT and CTB from pregnancies at 8–10 and 12–14 weeks' gestation. Gestational age and cell type differences were observed. EVT and CTB are major producers of angiogenic growth factors that likely contribute to placental vascular development and spiral artery remodeling.

Deficient activation and different expression of transforming growth factor-beta isoforms in active proliferative diabetic retinopathy and neovascular eye disease.

An increased expression and secretion of angiogenic growth factors was proposed to occur in proliferative diabetic retinopathy and other neovascularizing retinal diseases. However, a loss of anti-angiogenic factors also might promote retinal neovascularization. Therefore we investigated the active and latent vitreous levels of the subtypes of the endothelial anti-mitogen transforming growth factor-beta in vitreous of 58 patients. Four groups of patients were compared: Controls without retinal hypoxia, patients with quiescent and active proliferative diabetic retinopathy (PDR), and patients with severe retinal hypoxia resulting in rubeosis iridis. Whereas the amount of total TGF-beta in the four groups did not differ significantly, latent TGF-beta isoform expression showed complex alterations in ocular vitreous. Levels of active TGF-beta of patients with active PDR (79.5 +/- 28 pg/ml; n = 8) were decreased to 20% of the control levels (378 +/- 55 pg/ml; n = 12; p = 0.0005) and 25% of the mean concentration in quiescent PDR (346 +/- 64 pg/ml; n = 9; p = 0.0021). Levels in rubeosis (52 +/- 10 pg/ml; n = 10) did not differ significantly from those found in active PDR but were decreased to 15% of those in patients with quiescent PDR (p = 0.0004). Furthermore a highly significant inverse correlation between active TGF-beta and alpha2-antiplasmin, a liver produced inhibitor of the activation of TGF-beta by plasmin was noted (r = -0.59; n = 28; p = 0.001). We conclude that deficient activation of TGF-beta occurs in active proliferative diabetic retinopathy and in hypoxic angiogenesis most likely as a consequence of a blood retina barrier breakdown and influx of alpha2-antiplasmin from serum. The disinhibition of endothelial cell proliferation may be a central component in the process of neovascularization.

Locally Delivered Growth Factor Enhances the Angiogenic Efficacy of Adipose-Derived Stromal Cells Transplanted to Ischemic Limbs

Ischemia is a potentially fatal medical event that is associated with as many as 30% of all deaths. Stem cell therapy offers significant therapeutic promise, but poor survival following transplantation to ischemic tissue limits its efficacy. Here we demonstrate that nanosphere-mediated growth factor delivery can enhance the survival of transplanted human adipose-derived stromal cells (hADSCs) and secretion of human angiogenic growth factors per cell, and substantially improve therapeutic efficacy of hADSCs. In vitro, in hypoxic (1% oxygen) and serum-deprived conditions that simulate in vivo ischemia, fibroblast growth factor-2 (FGF2) significantly reduced hADSC apoptosis and enhanced angiogenic growth factor secretion. In vivo, hADSCs delivered intramuscularly into ischemic hind limbs in combination with FGF2 resulted in significant improvements in limb survival and blood perfusion, as well as survival of the transplanted hADSCs and secretion of human angiogenic growth factors (i.e., vascular endothelial growth factor, hepatocyte growth factor, and FGF2). Interestingly, the majority of transplanted hADSCs were localized adjacent to the microvessels rather than being incorporated into them, suggesting that their major contribution to angiogenesis might be to increase paracrine secretion of angiogenic growth factors. This study demonstrates the potential of hADSCs in combination with growth factors for use in the treatment of ischemia.

Fibroblastic response to treatment with different preparations rich in growth factors

Preparations rich in growth factors (PRGF) release them plus bioactive proteins at localized sites, with the aim of triggering healing and regenerative processes. The prevailing paradigm suggests that their influence on proliferation, angiogenesis and the extracellular matrix synthesis is minimal. However, variations in their composition and impact on different cell phenotypes have not been examined. Sixteen fibroblast cultures obtained from three different anatomical sites (skin, synovium and tendon) of 16 donors were exposed to the molecular pool released from PRGF scaffolds, with increasing amounts of platelets. We evaluated cell proliferation, secretion of angiogenic growth factors (VEGF and HGF), synthesis of type I collagen and hyaluronic acid (HA), considering platelet dose and anatomical origin of the cells. Activity of transforming growth factor-beta (TGF-beta) in type I procollagen and HA synthesis was examined by adding exogenous TGF-beta to plasma preparations. All plasma preparations induced a significant proliferative response compared to non-stimulated cells (P < 0.05). Maximum proliferation rate was obtained with PRGF with 2-fold or 4-fold platelet concentration. Exposure to PRGF stimulated VEGF synthesis exclusively in tendon cells (P < 0.05), which also exhibited a different pattern of HGF production (P < 0.05). PRGF enhanced HA synthesis (P < 0.05), but did not alter collagen I production. Platelet-secreted TGF-beta may be involved in HA, but not in type I procollagen synthesis. Optimizing composition and use of platelet-rich products is crucial to enhancing the therapeutic potential of this technology. Our data show that the biological effects of PRGF may depend on concentration of platelets and on the anatomical source of the cells.

Thrombin increases inflammatory cytokine and angiogenic growth factor secretion in human adipose cells in vitro

BackgroundAbdominal obesity is associated with pro-thrombotic and inflammatory states. Therefore, the purpose of this study was to examine the expression of thrombin receptors (PAR1 and PAR4) human adipose tissue and whether thrombin stimulates an inflammatory cytokine and growth factor profile in human adipose tissue.MethodsHuman adipose tissue, isolated preadipocytes and differentiated adipocytes were used in this study. PAR1 and PAR4 mRNA and protein were detected by RT-PCR and immunoblot analysis in both adipose tissue and adipose microvessels. In separate studies, IL-1β, IL-6, MCP-1, TNF-α, IL-10, FGF-2, VEGF, and PDGF production were measured from adipose tissue (n = 5), adipocytes (n = 5), and preadipocytes (n = 3) supernatants with and without thrombin (1 or 10 U/ml; 24 hrs) treatment.ResultsThrombin increased cytokine secretion of IL-1β, IL-6, MCP-1 and TNF-α and growth factor secretion of VEGF from adipocytes along with MCP-1 and VEGF from preadipocytes. The direct thrombin inhibitor lepirudin given in conjunction with thrombin prevented the thrombin-mediated increase in cytokine and growth factor secretion.ConclusionHere we show that thrombin PAR1 and PAR4 receptors are present and that thrombin stimulates inflammatory cytokine generation and growth factor release in human adipose tissue and cells in vitro. These data suggest that thrombin may represent a molecular link between obesity and associated inflammation.

Leptin and EPCs in Arterial Injury

See related article, pages 536–544 Endothelial progenitor cells (EPCs) were introduced to a broad scientific readership in 1997 by Asahara et al, who demonstrated that CD34+ cells from the peripheral blood can adopt an endothelial cell-like phenotype in vitro.1 This culture-modified cell type (subsequently also termed endothelial outgrowth cells) improved ischemic neovascularization after intravenous transfusion.2 The prospect of ameliorating tissue ischemia by ex vivo–expanded autologous angioblasts resulted in extensive research activities, including the therapeutic application in patients with myocardial ischemia.3 However, the results are still conflicting, which is at least partially attributable to the fact that EPCs comprise a heterogenous pool of subpopulations originating from distinct sources and displaying diverse phenotypes.4 For instance, the common characterization of EPCs as CD34+CD133+VEGF-R2+ by flow cytometry has been recently questioned in different studies showing that only CD133−CD45− cells differentiate into endothelial cells.5,6 Early outgrowth of endothelial-like cells from mononuclear cells cultured for 5 to 7 days in the presence of endothelial growth factors represent a monocyte-like subtype with low proliferative capacity secreting high …

Cardiac growth and angiogenesis coordinated by intertissue interactions

Cardiac hypertrophy and angiogenesis are coordinately regulated during physiological or adaptive cardiac growth, and disruption of the balanced growth and angiogenesis leads to contractile dysfunction and heart failure. Coordination of growth and angiogenesis is in part mediated by the secretion of angiogenic growth factors from myocytes in response to hypertrophic stimuli, which enables the vasculature to "catch up" to the growth of the myocardium. In this issue of the JCI, two studies provide novel insights into the regulatory mechanisms of cardiac growth and coronary angiogenesis. Heineke et al. demonstrate that GATA4 acts as a stress-responsive transcription factor in murine cardiac myocytes that induces the expression of angiogenic growth factors (see the related article beginning on page 3198). Tirziu et al. show that enhanced coronary angiogenesis per se leads to hypertrophic growth of myocytes through a nitric oxide-dependent mechanism (see the related article beginning on page 3188). These studies, together with previous reports, suggest the existence of reciprocal signals between the myocardium and the vasculature that promote the growth of each other in a paracrine fashion.

Endothelial Progenitor Cells: From Bench to Bedside

Strong evidence suggests that bone marrow-derived cells play a role in physiological and pathological blood vessel growth in the adult, both by augmenting angiogenesis through the secretion of angiogenic growth factors and by providing a rich source of progenitor cells that can differentiate into mature vascular endothelial cells. This is a true paradigm shift, since adult neovascularization processes were thought to be limited to angiogenesis. The cells that are critical to postnatal blood vessel growth - endothelial progenitor cells - may be analogous to the embryonic angioblast, in that they can circulate, proliferate and participate in the development of vascular networks by differentiating in situ, probably via the formation of cell clusters into mature endothelial cells. Therefore, initial reports have seen analogs to the process of vasculogenesis in the embryo, where the de novo synthesis of vessels occurs through the formation of blood island-like clusters, which subsequently connect and eventually form systemic vasculature. Recent work implicates precursors of endothelial cells in such processes as myocardial ischemia and infarction, limb ischemia, wound healing, atherosclerosis, endogenous endothelial repair and tumor vascularization. These new insights into the vascular biology of endothelial regeneration and repair led to the development of new cell therapeutic strategies to enhance adult neovascularization and re-endothelialization in ischemic cardiovascular diseases.

Bioactive Glass Stimulates the Secretion of Angiogenic Growth Factors and Angiogenesis in Vitro

Neovascularization of tissue-engineered constructs remains a limiting factor for the engineering of larger tissue constructs. Attempts to stimulate neovascularization, using recombinant protein or gene transfer of angiogenic growth factors, have been proposed; however, these approaches have been associated with problems regarding the delivery and duration of exposure of the growth factor. This study was performed to determine the ability of biologically active glass to stimulate the secretion of angiogenic growth factors from human stromal cells and subsequent angiogenesis. CCD18Co human fibroblasts were cultured on tissue culture surfaces coated with specific quantities of 45S5 Bioglass particles. At 24-, 48-, and 72-h intervals the gene expression of vascular endothelial growth factor (VEGF) and the protein secretion of VEGF and basic fibroblast growth factor (bFGF) from fibroblasts were measured. The effect of conditioned medium collected from Bioglass-stimulated fibroblasts on human dermal microvascular endothelial cells was assessed using in vitro angiogenesis assays. Results showed that surfaces coated with Bioglass produced a significant increase in the secretion of VEGF and bFGF. Conditioned medium from stimulated fibroblasts significantly increased the proliferation of human dermal microvascular endothelial cells and induced a significant increase in the formation of anastomosed networks of human endothelial cell tubules. It is concluded that the ability of 45S5 Bioglass to stimulate the release of angiogenic growth factors and to promote angiogenesis provides a novel alternative approach for stimulating neovascularization of tissue-engineered constructs.

The Cyclooxygenase Inhibitor Flurbiprofen Reduces Radiation‐Induced Angiogenic Growth Factor Secretion of Squamous Cell Carcinoma Cell Lines

Surgical intervention and radiotherapy still represent the gold standard in the therapy of head and neck squamous cell carcinoma (SCC), although often with unsatisfactory results. Radiation might induce the expression and secretion of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) by unknown mechanisms. These two highly active proangiogenic and cytoprotective factors might contribute to a limited therapeutic success by promoting revascularization and cytoprotection of the radiated tumor. The aim of the present study was to analyze the potential of the cyclooxygenase inhibitor flurbiprofen to reduce radiation-induced increase of VEGF and bFGF secretion of tumor cells. We analyzed the expression of VEGF and bFGF at 72 h after radiation with 30 Gy in four SCC cell lines (De-pt, Hun, Lau, and A549) in cell culture with or without added flurbiprofen. Controls were not exposed to radiation and were analyzed at the same time after culture in the same media. We observed increased VEGF levels in all and increased bFGF levels in three of four lines after radiation. In irradiated cultures with flurbiprofen, VEGF was reduced between 13% and 26% and bFGF was reduced between 84% and 93% compared with radiated cultures without flurbiprofen. We found no reduction of VEGF and bFGF secretion in the unirradiated cultures despite added flurbiprofen. We conclude that flurbiprofen is able to alter the radiation-induced secretion of these two growth factors and might be useful in decreasing the resistance of SCC to radiation.

The small GTPase Rac1 links the Kaposi sarcoma–associated herpesvirus vGPCR to cytokine secretion and paracrine neoplasia

AbstractKaposi sarcoma (KS) is a multifocal angioproliferative neoplasm strictly dependent on angiogenic growth factors and cytokines and invariably associated with infection by the Kaposi sarcoma–associated herpesvirus (KSHV or HHV8). A G protein–coupled receptor encoded by KSHV (vGPCR) is able to initiate KS-like tumors when targeted to the vascular endothelium of mice. Analogous to human KS, vGPCR sarcomagenesis involves the paracrine secretion of angiogenic growth factors and proinflammatory molecules from vGPCR-expressing cells. Here we demonstrate that vGPCR up-regulates expression and secretion of critical KS cytokines by stimulating key transcription factors, including nuclear factor–κB (NF-κB), activator protein-1 (AP-1), and nuclear factor of activated T cells (NFAT), through the activation of the small G protein Rac1. Inhibition of Rac1 blocked vGPCR-induced transcription and secretion of KS cytokines, including interleukin-6 (IL-6), IL-8, and growth-regulated oncogene α (GROα), in vitro and reduced vGPCR tumorigenesis in vivo. Moreover, endothelial-specific infection with the constitutively active Rac1QL induced vascular lesions in mice that were remarkably similar to early vGPCR experimental lesions. These results identify Rac1 as a key mediator of vGPCR paracrine neoplasia, suggesting that this small G protein and its downstream effectors may represent suitable therapeutic targets for the treatment of KS.