Targeting IL-3Rα on tumor-derived endothelial cells blunts metastatic spread of triple-negative breast cancer via extracellular vesicle reprogramming.

The lack of approved targeted therapies highlights the need for new treatments for triple-negative breast cancer (TNBC) patients. Interleukin-3 (IL-3) acts as an autocrine factor for tumor-endothelial cells (TEC), and exerts pro-angiogenic paracrine action via extracellular vesicles (EVs). IL-3Rα blockade on TEC changes TEC-EV (anti-IL-3R-EV) microRNA (miR) content and promotes the regression of established vessels. As TEC is the doorway for "drug" entry into tumors, we aimed to assess whether IL-3R blockade on TEC impacts tumor progression via its unique EV cargo. First, the expression of IL-3Rα was evaluated in 27 human TNBC samples. It was noticed that, besides TEC and inflammatory cells, tumor cells from 55.5% of the human TNBC samples expressed IL-3Rα. Using human TNBC cell lines for in vitro studies, we found that, unlike native TEC-EVs (nEVs), anti-IL-3R-EVs increase apoptosis and reduced cell viability and migration. In vivo, anti-IL-3R-EV treatment induced vessel regression in established tumors formed of MDA-MB-231 cells, decreased Vimentin, ß-catenin, and TWIST1 expression, almost abolished liver and lung metastases from primary tumors, and reduced lung metastasis generated via the intravenous injection of MDA-MB-231 cells. nEVs depleted of miR-24-3p (antago-miR-24-3p-EVs) were effective as anti-IL-3R-EVs in downregulating TWIST1 and reducing metastatic lesions in vivo. Consistent with network analyses of miR-24-3p gene targeting, anti-IL-3R-EVs and antago-miR-24-3p-EVs upregulate SPRY2 in MDA-MB-231 cells. Finally, SPRY2 silencing prevented anti-IL-3R-EV and antago-miR-24-3p-EV-mediated apoptotic cues.Overall, these data provide the first evidence that IL-3Rα is highly expressed in TNBC cells, TEC, and inflammatory cells, and that IL-3Rα blockade on TEC impacts tumor progression.

PDGF-BB Carried by Endothelial Cell-Derived Extracellular Vesicles Reduces Vascular Smooth Muscle Cell Apoptosis in Diabetes.

Endothelial cell-derived extracellular vesicles (CD31EVs) constitute a new entity for therapeutic/prognostic purposes. The roles of CD31EVs as mediators of vascular smooth muscle cell (VSMC) dysfunction in type 2 diabetes (T2D) are investigated herein. We demonstrated that, unlike serum-derived extracellular vesicles in individuals without diabetes, those in individuals with diabetes (D CD31EVs) boosted apoptosis resistance of VSMCs cultured in hyperglycemic condition. Biochemical analysis revealed that this effect relies on changes in the balance between antiapoptotic and proapoptotic signals: increase of bcl-2 and decrease of bak/bax. D CD31EV cargo analysis demonstrated that D CD31EVs are enriched in membrane-bound platelet-derived growth factor-BB (mbPDGF-BB). Thus, we postulated that mbPDGF-BB transfer by D CD31EVs could account for VSMC resistance to apoptosis. By depleting CD31EVs of platelet-derived growth factor-BB (PDGF-BB) or blocking the PDGF receptor ß on VSMCs, we demonstrated that mbPDGF-BB contributes to D CD31EV-mediated bak/bax and bcl-2 levels. Moreover, we found that bak expression is under the control of PDGF-BB-mediated microRNA (miR)-296-5p expression. In fact, while PDGF-BB treatment recapitulated D CD31EV-mediated antiapoptotic program and VSMC resistance to apoptosis, PDGF-BB-depleted CD31EVs failed. D CD31EVs also increased VSMC migration and recruitment to neovessels by means of PDGF-BB. Finally, we found that VSMCs, from human atherosclerotic arteries of individuals with T2D, express low bak/bax and high bcl-2 and miR-296-5p levels. This study identifies the mbPDGF-BB in D CD31EVs as a relevant mediator of diabetes-associated VSMC resistance to apoptosis.

Stem Cell-Derived, microRNA-Carrying Extracellular Vesicles: A Novel Approach to Interfering with Mesangial Cell Collagen Production in a Hyperglycaemic Setting.

Extracellular vesicles (EVs) that are derived from stem cells are proving to be promising therapeutic options. We herein investigate the therapeutic potential of EVs that have been derived from different stem cell sources, bone-marrow (MSC) and human liver (HLSC), on mesangial cells (MCs) exposed to hyperglycaemia. By expressing a dominant negative STAT5 construct (ΔNSTAT5) in HG-cultured MCs, we have demonstrated that miR-21 expression is under the control of STAT5, which translates into Transforming Growth Factor beta (TGFß) expression and collagen production. A number of approaches have been used to show that both MSC- and HLSC-derived EVs protect MCs from HG-induced damage via the transfer of miR-222. This resulted in STAT5 down-regulation and a decrease in miR-21 content, TGFß expression and matrix protein synthesis within MCs. Moreover, we demonstrate that changes in the balance between miR-21 and miR-100 in the recipient cell, which are caused by the transfer of EV cargo, further contribute to providing beneficial effects. Interestingly, these effects were only detected in HG-cultured cells. Finally, it was found that HG reduced the expression of the nuclear encoded mitochondrial electron transport chain (ETC) components, CoxIV. It is worth noting that EV administration can rescue CoxIV expression in HG-cultured MCs. These results thus demonstrate that both MSC- and HLSC-derived EVs transfer the machinery needed to preserve MCs from HG-mediated damage. This occurs via the horizontal transfer of functional miR-222 which directly interferes with damaging cues. Moreover, our data indicate that the release of EV cargo into recipient cells provides additional therapeutic advantages against harmful mitochondrial signals.

Activated Stat5 trafficking Via Endothelial Cell-derived Extracellular Vesicles Controls IL-3 Pro-angiogenic Paracrine Action.

Soluble factors and cell-derived extracellular vesicles (EVs) control vascular cell fate during inflammation. The present study investigates the impact of Interleukin 3 (IL-3) on EV release by endothelial cells (ECs), the mechanisms involved in EV release and paracrine actions. We found that IL-3 increases EV release, which is prevented by IL-3Ralpha blockade. EVs released upon IL-3 stimulation were able to induce pro-angiogenic signals as shown by chromatin immunoprecipitation (ChIP) assay performed on the promoter region of cyclin D1 and tridimensional tube-like structure formation. We herein demonstrate that these effects rely on the transfer of miR-126-3p, pre-miR-126 and, more importantly, of activated signal transduction and activator of transcription 5 (pSTAT5) from IL-3-EV cargo into recipient ECs. We show, using the dominant negative form (ΔN)STAT5 and an activated STAT5 (1*6STAT5) constructs, that STAT5 drives IL-3-mediated EV release, miR-126-3p and pSTAT5 content. Finally, using EVs recovered from ΔNSTAT5 expressing ECs, we provide evidence that miR-126-3p and pSTAT5 trafficking is relevant for IL-3-mediated paracrine pro-angiogenic signals. These results indicate that IL-3 regulates EC-EV release, cargo and IL-3 angiogenic paracrine action via STAT5. Moreover, these results provide evidence that EC-derived IL-3-EVs can serve as pro-angiogenic clinical delivery wound healing devices.

Unacylated ghrelin induces oxidative stress resistance in a glucose intolerance and peripheral artery disease mouse model by restoring endothelial cell miR-126 expression.

Reactive oxygen species (ROS) are crucial in long-term diabetes complications, including peripheral artery disease (PAD). In this study, we have investigated the potential clinical impact of unacylated ghrelin (UnAG) in a glucose intolerance and PAD mouse model. We demonstrate that UnAG is able to protect skeletal muscle and endothelial cells (ECs) from ROS imbalance in hind limb ischemia-subjected ob/ob mice. This effect translates into reductions in hind limb functional impairment. We show that UnAG rescues sirtuin 1 (SIRT1) activity and superoxide dismutase-2 (SOD-2) expression in ECs. This leads to SIRT1-mediated p53 and histone 3 lysate 56 deacetylation and results in reduced EC senescence in vivo. We demonstrate, using small interfering RNA technology, that SIRT1 is also crucial for SOD-2 expression. UnAG also renews micro-RNA (miR)-126 expression, resulting in the posttranscriptional regulation of vascular cell adhesion molecule 1 expression and a reduced number of infiltrating inflammatory cells in vivo. Loss-of-function experiments that target miR-126 demonstrate that miR-126 also controls SIRT1 and SOD-2 expression, thus confirming its role in driving UnAG-mediated EC protection against ROS imbalance. These results indicate that UnAG protects vessels from ROS imbalance in ob/ob mice by rescuing miR-126 expression, thus emphasizing its potential clinical impact in avoiding limb loss in PAD.

miR-221/222 control luminal breast cancer tumor progression by regulating different targets.

α6ß4 integrin is an adhesion molecule for laminin receptors involved in tumor progression. We present a link between ß4 integrin expression and miR-221/222 in the most prevalent human mammary tumor: luminal invasive carcinomas (Lum-ICs). Using human primary tumors that display different ß4 integrin expression and grade, we show that miR-221/222 expression inversely correlates with tumor proliferating index, Ki67. Interestingly, most high-grade tumors express ß4 integrin and low miR-221/222 levels. We ectopically transfected miR-221/222 into a human-derived mammary tumor cell line that recapitulates the luminal subtype to investigate whether miR-221/222 regulates ß4 expression. We demonstrate that miR-221/222 overexpression results in ß4 expression downregulation, breast cancer cell proliferation, and invasion inhibition. The role of miR-221/222 in driving ß4 integrin expression is also confirmed via mutating the miR-221/222 seed sequence for ß4 integrin 3'UTR. Furthermore, we show that these 2 miRNAs are also key breast cancer cell proliferation and invasion regulators, via the post-transcriptional regulation of signal transducer and activator of transcription 5A (STAT5A) and of a disintegrin and metalloprotease-17 (ADAM-17). We further confirm these data by silencing ADAM-17, using a dominant-negative or an activated STAT5A form. miR-221/222-driven ß4 integrin, STAT5A, and ADAM-17 did not occur in MCF-10A cells, denoted "normal" breast epithelial cells, indicating that the mechanism is cancer cell-specific.   These results provide the first evidence of a post-transcriptional mechanism that regulates ß4 integrin, STAT5A, and ADAM-17 expression, thus controlling breast cancer cell proliferation and invasion. Pre-miR-221/222 use in the aggressive luminal subtype may be a powerful therapeutic anti-cancer strategy.

DNA vaccination against membrane-bound Kit ligand: a new approach to inhibiting tumour growth and angiogenesis.

A functional c-Kit/Kit ligand (KitL) signalling network is required for tumour angiogenesis and growth, and therefore the c-Kit/KitL system might well be a suitable target for the cancer immunotherapy approach. We herein describe a strategy that targets membrane-bound KitL (mbKitL) via DNA vaccination. The vaccination procedure generated antibodies which are able to detect mbKitL on human tumour endothelial cells (TECs) and on the breast cancer cell line: TSA. DNA vaccination, interferes with tumour vessel formation and transplanted tumour growth in vivo. Histological analysis demonstrates that, while tumour cell proliferation and vessel stabilisation are impaired, vessel permeability is increased in mice that produce mbKitL-targeting antibodies. We also demonstrate that vessel stabilisation and tumour growth require Akt activation in endothelial cells but not in pericytes. Moreover, we found that regulatory T cells (Treg) and tumour infiltrating inflammatory cells, involved in tumour growth and angiogenesis, were reduced in number in the tumour microenvironment of mice that generate anti-mbKitL antibodies. These data provide evidence that mbKitL targeted vaccination is an effective means of inhibiting tumour angiogenesis and growth.

Unacylated ghrelin promotes skeletal muscle regeneration following hindlimb ischemia via SOD-2-mediated miR-221/222 expression.

BACKGROUND: Surgical treatment of peripheral artery disease, even if successful, does not prevent reoccurrence. Under these conditions, increased oxidative stress is a crucial determinant of tissue damage. Given its reported antioxidant effects, we investigated the potential of unacylated-ghrelin (UnAG) to reduce ischemia-induced tissue damage in a mouse model of peripheral artery disease. METHODS AND RESULTS: We show that UnAG but not acylated ghrelin (AG) induces skeletal muscle regeneration in response to ischemia via canonical p38/mitogen-actived protein kinase signaling UnAG protected against reactive oxygen species-induced cell injuries by inducing the expression of superoxide dismutase-2 (SOD-2) in satellite cells. This led to a reduced number of infiltrating CD68(+) cells and was followed by induction of the myogenic process and a reduction in functional impairment. Moreover, we found that miR-221/222, previously linked to muscle regeneration processes, was up-regulated and negatively correlated with p57(Kip2) expression in UnAG-treated mice. UnAG, unlike AG, promoted cell-cycle entry in satellite cells of mice lacking the genes for ghrelin and its receptor (GHSR1a). UnAG-induced p38/mitogen-actived protein kinase phosphorylation, leading to activation of the myogenic process, was prevented in SOD-2-depleted SCs. By siRNA technology, we also demonstrated that SOD-2 is the antioxidant enzyme involved in the control of miR-221/222-driven posttranscriptional p57(Kip2) regulation. Loss-of-function experiments targeting miR-221/222 and local pre-miR-221/222 injection in vivo confirmed a role for miR-221/222 in driving skeletal muscle regeneration after ischemia. CONCLUSIONS: These results indicate that UnAG-induced skeletal muscle regeneration after ischemia depends on SOD-2-induced miR-221/222 expression and highlight its clinical potential for the treatment of reactive oxygen species-mediated skeletal muscle damage.

Increase of palmitic acid concentration impairs endothelial progenitor cell and bone marrow-derived progenitor cell bioavailability: role of the STAT5/PPARγ transcriptional complex.

Metabolic profiling of plasma nonesterified fatty acids discovered that palmitic acid (PA), a natural peroxisome proliferator-activated receptor γ (PPARγ) ligand, is a reliable type 2 diabetes biomarker. We investigated whether and how PA diabetic (d-PA) concentrations affected endothelial progenitor cell (EPC) and bone marrow-derived hematopoietic cell (BM-HC) biology. PA physiologic (n-PA) and d-PA concentrations were used. Proliferating cell nuclear antigen content and signal transducer and activator of transcription 5 (STAT5), PPARγ, cyclin D1, and p21(Waf) expression were evaluated. Small interfering RNA technology, gene reporter luciferase assay, electrophoretic mobility shift assay, chromatin immunoprecipitation assay, and coimmunoprecipitation were exploited. In vivo studies and migration assays were also performed. d-PA, unlike n-PA or physiological and diabetic oleic and stearic acid concentrations, impaired EPC migration and EPC/BM-HC proliferation through a PPARγ-mediated STAT5 transcription inhibition. This event did not prevent the formation of a STAT5/PPARγ transcriptional complex but was crucial for gene targeting, as p21(Waf) gene promoter, unlike cyclin D1, was the STAT5/PPARγ transcriptional target. Similar molecular events could be detected in EPCs isolated from type 2 diabetic patients. By expressing a constitutively activated STAT5 form, we demonstrated that STAT5 content is crucial for gene targeting and EPC fate. Finally, we also provide in vivo data that d-PA-mediated EPC dysfunction could be rescued by PPARγ blockade. These data provide first insights on how mechanistically d-PA drives EPC/BM-HC dysfunction in diabetes.

microRNA-222 controls neovascularization by regulating signal transducer and activator of transcription 5A expression.

OBJECTIVE: Inflammatory stimuli released into atherosclerotic plaque microenvironment regulate vessel formation by modulating gene expression and translation. microRNAs are a class of short noncoding RNAs, acting as posttranscriptional regulators of protein-coding genes involved in various biological processes, including vascular cell biology. Among them, microRNA-221/222 (miR-221/222) seem to negatively modulate vascular remodeling by targeting different target genes. Here, we investigated their potential contribution to inflammation-mediated neovessel formation. METHODS AND RESULTS: We used quantitative real-time RT-PCR amplification to analyze expression of 7 microRNAs previously linked to vascular biology, such as miR-17-5p, miR-21, miR-126, miR-210, miR-221, miR-222, and miR-296 and found high levels of expression for all of them in quiescent endothelial cells. However, miR-126, miR-221, miR-222, and miR-296 turned out to be down-modulated in endothelial cells exposed to inflammatory stimuli. Applying a gain-of-function approach, we demonstrated that, among them, only miR-222 was involved in inflammation-mediated vascular remodeling. In addition, we identified signal transducer and activator of transcription 5A (STAT5A) as a bona fide target of miR-222 and observed that miR-222 negatively correlated with STAT5A expression in human endothelial cells from advanced neovascularized atherosclerotic lesions. CONCLUSIONS: We identified STAT5A as a novel miR-222 target, and this finding opens up new perspectives for treatment of vascular diseases.

Unacylated ghrelin rescues endothelial progenitor cell function in individuals with type 2 diabetes.

OBJECTIVE: Acylated ghrelin (AG) is a diabetogenic and orexigenic gastric polypeptide. These properties are not shared by the most abundant circulating form, which is unacylated (UAG). An altered UAG/AG profile together with an impairment of circulating endothelial progenitor cell (EPC) bioavailability were found in diabetes. Based on previous evidence for the beneficial cardiovascular effects of AG and UAG, we investigated their potential to revert diabetes-associated defects. RESEARCH DESIGN AND METHODS: Healthy human subjects, individuals with type 2 diabetes, and ob/ob mice were AG or UAG infused. EPC mobilization in patients and mice was evaluated, and the underlying molecular mechanisms were investigated in bone marrow stromal cells. Recovered EPCs were also evaluated for the activity of senescence regulatory pathways and for NADPH oxidase activation by knocking down p47(phox) and Rac1. Finally, UAG modulation of human EPC vasculogenic potential was investigated in an in vivo mouse model. RESULTS: Neither AG nor UAG had any effect in healthy subjects. However, systemic administration of UAG, but not AG, prevented diabetes-induced EPC damage by modulating the NADPH oxidase regulatory protein Rac1 and improved the vasculogenic potential both in individuals with type 2 diabetes and in ob/ob mice. In addition, unlike AG, UAG facilitated the recovery of bone marrow EPC mobilization. Crucial to EPC mobilization by UAG was the rescue of endothelial NO synthase (eNOS) phosphorylation by Akt, as UAG treatment was ineffective in eNOS knockout mice. Consistently, EPCs expressed specific UAG-binding sites, not recognized by AG. CONCLUSIONS: These data provide the rationale for clinical applications of UAG in pathologic settings where AG fails.

Establishment and characterization of a human retinal pericyte line: a novel tool for the study of diabetic retinopathy.

Loss of pericytes from retinal microvessels is one of the key events in the natural history of diabetic retinopathy. Cultured human retinal pericytes would constitute an extremely useful tool for the study of the early events in the pathogenesis of this complication, but, due to legal and ethical issues, pericytes of animal origin have been mostly used so far for in vitro assays. We aimed at establishing an immortalized human retinal pericyte (HRP) line, as a species-specific model to investigate the pericyte-related aspects of diabetic retinopathy. Primary human retinal pericytes (WT-HRP) were immortalized through electroporation with a plasmid vector containing the Bmi-1 oncogene that induces telomerase activity, resulting in the establishment of a permanent pericyte line (Bmi-HRP), which showed telomerase activity and facilitated propagation. The immortalized cells were characterized for typical pericyte morphology and marker expression. Immunofluorescence studies demonstrated that Bmi-HRP maintain the same morphology and express the typical markers of wild-type pericytes. The response of the cell line to high glucose damaging stimulus was also evaluated, as senescence-associated beta-galactosidase activity and cell proliferation and a clear negative effect of high glucose on Bmi-HRP proliferation and senescence, in line with the characteristic response of wild-type cells, was observed. The combination of infinite proliferation capability and stable differentiation potential makes our Bmi-HRP line a promising candidate model to study pathogenic mechanisms and therapeutic applications in diabetic retinopathy.

Formation of STAT5/PPARgamma transcriptional complex modulates angiogenic cell bioavailability in diabetes.

OBJECTIVE: Circulating angiogenic cells (CACs) expansion is a multistage process requiring sequential activation of transcriptional factors, including STAT5. STAT5, in concert with peroxisome proliferator-activated receptors (PPARs), seems to induce discrete biological responses in different tissues. In the present study we investigated the role of STAT5 and PPARgamma in regulating CAC expansion in normal and diabetic settings. METHODS AND RESULTS: Normal and diabetic CACs were used. siRNA technology, EMSA, and chromatin immunoprecipitation (ChIP) assay as well as site-directed mutagenesis of the STAT5 response element in the PPARgamma promoter enabled us to demonstrate that STAT5 transcriptional activity controls PPARgamma expression. Moreover, FACS analysis, coimmunoprecipitation experiments, and ChIP assay revealed that a STAT5/PPARgamma transcriptional complex controls cyclin D1 expression and CAC progression into the cell-cycle. Conversely, PPARgamma agonists, by preventing the expression of STAT5 and the formation of the STAT5/PPARgamma heterodimeric complex failed to promote CAC expansion. Finally, we demonstrated that diabetic CAC functional capability can be recovered by molecules able to activate the STAT5/PPARgamma transcriptional complex. CONCLUSIONS: Our data identify the STAT5/PPARgamma heterodimers as landmark of CAC expansion and provide evidences for a mechanism that partially rescues CAC bioavailability in diabetic setting.

Interleukin-3 promotes expansion of hemopoietic-derived CD45+ angiogenic cells and their arterial commitment via STAT5 activation.

Interleukin-3 (IL-3) released by infiltrating inflammatory cells in different pathologic settings contributes to organ and tumor angiogenesis. Here we demonstrate that IL-3 expands a subset of CD45+ circulating angiogenic cells clonally derived from the hemopoietic progenitors. Moreover, CD45+ cells exposed to IL-3 acquire arterial specification and contribute to the formation of vessels in vivo. Depletion of signal transducer and activator of transcription 5 (STAT5) provides evidence that IL-3-mediated cell expansion and arterial morphogenesis rely on STAT5 activation. In addition, by means of Tie2-transgenic mice, we demonstrate that STAT5 also regulates IL-3-induced expansion and arterial specification of bone marrow-derived CD45+ cells. Thus, our data provide the first evidence that, in inflammatory microenvironments containing IL-3, angiogenic cells derived from hemopoietic precursors can act as adult vasculogenic cells. Moreover, the characterization of the signaling pathway regulating these events provides the rationale for therapeutically targeting STAT5 in these pathologic settings.

Oxidative stress-mediated mesangial cell proliferation requires RAC-1/reactive oxygen species production and beta4 integrin expression.

Lipid abnormalities and oxidative stress, by stimulating mesangial cell (MC) proliferation, can contribute to the development of diabetes-associated renal disease. In this study we investigated the molecular events elicited by oxidized low density lipoproteins (ox-LDL) in MC. We demonstrate that in MC cultured in the presence of ox-LDL, survival and mitogenic signals on Akt and Erk1/2 MAPK pathways are induced, respectively. Moreover, as shown by the expression of the dominant negative Rac-1 construct, we first report that ox-LDL-mediated cell survival and cell cycle progression depend on Rac-1 GTPase-mediated reactive oxygen species production and on epidermal growth factor receptor transactivation. By silencing Akt and blocking Erk1/2 MAPK pathways, we also demonstrate that these signals are downstream to Rac-1/reactive oxygen species production and epidermal growth factor receptor activation. Finally, by endogenous depletion of beta4 integrin, expressed in MC, we provide evidence that the expression of this adhesion molecule is essential for ox-LDL-mediated MC dysfunction. Our data identify a novel signaling pathway involved in oxidative stress-induced diabetes-associated renal disease and provide the rationale for therapeutically targeting beta4 integrin.

C-KIT, by interacting with the membrane-bound ligand, recruits endothelial progenitor cells to inflamed endothelium.

We investigated the role of c-Kit and the membrane-bound ligand (mbKitL) in endothelial progenitor cell (EPC) recruitment by microvascular endothelial cells (ECs). We demonstrated that inflammatory activation induced the expression of the mbKitL on ECs both in vitro and in vivo, and that recruitment of EPCs depended on c-Kit/mbKitL interaction. Depletion of endogenous c-Kit or inhibition of c-Kit enzymatic activity by imatinib mesylate prevented adhesion of EPCs to activated ECs both in vitro and in vivo, indicating that a functional c-Kit on EPCs is essential. We also demonstrate that Akt was the downstream molecule regulating cell adhesion. A potential role of the c-Kit/mbKitL interaction in pathological settings is sustained by the expression of the mbKitL on ECs lining intraplaque neovessels. Thus, our results provide new insights into the mechanisms underlying EPC recruitment and the bases for novel strategies to hinder pathological angiogenesis.

p53 Mediates the accelerated onset of senescence of endothelial progenitor cells in diabetes.

Adverse metabolic factors, including oxidized small and dense low density lipoprotein (ox-dmLDL) can contribute to the reduced number and the impaired functions of circulating endothelial progenitors (EPC) in diabetic patients. To elucidate the molecular mechanisms involved, EPC from normal donors were cultured in the presence of ox-dmLDL. Under these experimental conditions EPC undergo to senescent-like growth arrest. This effect is associated with Akt activation, p21 expression, p53 accumulation, and retinoblastoma protein dephosphorylation and with a reduced protective effect against oxidative damage. Moreover, depletion of endogenous p53 expression by small interfering RNA demonstrates that the integrity of this pathway is essential for senescence to occur. Activation of the Akt/p53/p21 signaling pathway and accelerated onset of senescence are also detectable in EPC from diabetic patients. Finally, diabetic EPC depleted of endogenous p53 do not undergo to senescence-growth arrest and acquire the ability to form tube-like structures in vitro. These observations identify the activation of the p53 signaling pathway as a crucial event that can contribute to the impaired neovascularization in diabetes.

The interaction between KDR and interleukin-3 receptor (IL-3R) beta common modulates tumor neovascularization.

As vascular endothelial growth factor (VEGF), interleukin-3 (IL-3), released into the tumor microenvironment stimulates motogenic and mitogenic activity of normal and transformed cells. In the present study, we investigate the effects of IL-3 and VEGF on neoplastic vascular growth. Engagement of IL-3 receptor beta common (IL-3R beta c) contributes to both IL-3- and VEGF-induced Rac1 activation, cell migration and in vitro tube-like structure formation as shown by the expression of the dominant-negative IL-3R beta c construct (Delta455). In normal and transformed endothelial cells (EC) as well as in HEK 293 cells expressing KDR and IL-3R, VEGF and IL-3 treatment induces the formation of a KDR/IL-3R beta c complex. Moreover, as shown by the IL-3R Delta455 mutant or by the kinase dead KDR, functional receptors are required for this interaction. Consistent with the contribution of IL-3R beta c in both IL-3- and VEGF-mediated angiogenic signal, a reduced number of vessels inside tumors are found in mice injected with cells expressing the IL-3R Delta455 mutant. Thus, these findings provide a novel mechanism through which IL-3 and VEGF support cell survival and tumor neovascularization.

{beta}1 Integrin and IL-3R coordinately regulate STAT5 activation and anchorage-dependent proliferation.

We previously demonstrated that integrin-dependent adhesion activates STAT5A, a well known target of IL-3-mediated signaling. Here, we show that in endothelial cells the active beta1 integrin constitutively associates with the unphosphorylated IL-3 receptor (IL-3R) beta common subunit. This association is not sufficient for activating downstream signals. Indeed, only upon fibronectin adhesion is Janus Kinase 2 (JAK2) recruited to the beta1 integrin-IL-3R complex and triggers IL-3R beta common phosphorylation, leading to the formation of docking sites for activated STAT5A. These events are IL-3 independent but require the integrity of the IL-3R beta common. IL-3 treatment increases JAK2 activation and STAT5A and STAT5B tyrosine and serine phosphorylation and leads to cell cycle progression in adherent cells. Expression of an inactive STAT5A inhibits cell cycle progression upon IL-3 treatment, identifying integrin-dependent STAT5A activation as a priming event for IL-3-mediated S phase entry. Consistently, overexpression of a constitutive active STAT5A leads to anchorage-independent cell cycle progression. Therefore, these data provide strong evidence that integrin-dependent STAT5A activation controls IL-3-mediated proliferation.