Endothelial Cell Morphogenesis Research Articles

Prevascularized hydrogels with mature vascular networks promote the regeneration of critical-size calvarial bone defects in vivo.

Adequate vascularization of scaffolds is a prerequisite for successful repair and regeneration of lost and damaged tissues. It has been suggested that the maturity of engineered vascular capillaries, which is largely determined by the presence of functional perivascular mural cells (or pericytes), plays a vital role in maintaining vessel integrity during tissue repair and regeneration. Here, we investigated the role of pericyte-supported-engineered capillaries in regenerating bone in a critical-size rat calvarial defect model. Prior to implantation, human umbilical vein endothelial cells and human bone marrow stromal cells (hBMSCs) were cocultured in a collagen hydrogel to induce endothelial cell morphogenesis into microcapillaries and hBMSC differentiation into pericytes. Upon implantation into the calvarial bone defects (8mm), the prevascularized hydrogels showed better bone formation than either untreated controls or defects treated with autologous bone grafts (positive control). Bone formation parameters such as bone volume, coverage area, and vascularity were significantly better in the prevascularized hydrogel group than in the autologous bone group. Our results demonstrate that tissue constructs engineered with pericyte-supported vascular capillaries may approximate the regenerative capacity of autologous bone, despite the absence of osteoinductive or vasculogenic growth factors.

Stromal cell identity modulates vascular morphogenesis in a microvasculature-on-a-chip platform.

Supportive stromal cells of mesenchymal origins regulate vascular morphogenesis in developmental, pathological, and regenerative contexts, contributing to vessel formation, maturation, and long-term stability, in part via the secretion of bioactive molecules. In this work, we adapted a microfluidic lab-on-a-chip system that enables the formation and perfusion of microvascular capillary beds with connections to arteriole-scale endothelialized channels to explore how stromal cell (SC) identity influences endothelial cell (EC) morphogenesis. We compared and contrasted lung fibroblasts (LFs), dermal fibroblasts (DFs), and bone marrow-derived mesenchymal stem cells (MSCs) for their abilities to support endothelial morphogenesis and subsequent perfusion of microvascular networks formed in fibrin hydrogels within the microfluidic device. We demonstrated that while all 3 SC types supported EC morphogenesis, LFs in particular resulted in microvascular morphologies with the highest total network length, vessel diameter, and vessel interconnectivity across a range of SC-EC ratio and density conditions. Not only did LFs support robust vascular morphology, but also, they were the only SC type to support functional perfusion of the resultant capillary beds. Lastly, we identified heightened traction stress produced by LFs as a possible mechanism by which LFs enhance endothelial morphogenesis in 3D compared to other SC types examined. This study provides a unique comparison of three different SC types and their role in supporting the formation of microvasculature that could provide insights for the choice of cells for vascular cell-based therapies and the regulation of tissue-specific vasculature.

Therapeutic Angiogenesis Using HGF Plasmid.

Hepatocyte growth factor (HGF) is secreted from stromal and mesenchymal cells, and its receptor cMet is expressed on various types of cells such as smooth muscle cells, fibroblast, and endothelial cells. HGF stimulates epithelial and endothelial cell proliferation, motility, and morphogenesis in a paracrine and autocrine manner, organizing multistep of angiogenesis in many organs. In addition, HGF is recognized as a potent anti-inflammatory and anti-fibrotic growth factor, which has been proved in several animal studies, including neointimal hyperplasia and acute myocardial infarction model in rodent. Thus, as compared to other angiogenic growth factors, HGF exerts multiple effects on ischemic tissues, accompanied by the regression of tissue inflammation and fibrosis. These data suggest the therapeutic potential of the HGF for peripheral artery disease as it being accompanied with chronic tissue inflammation and fibrosis. In the present narrative review, the pleiotropic action of the HGF that differentiates it from other angiogenic growth factors is discussed first, and later, outcomes of the human clinical study with gene therapy are overviewed.

Transient Support from Fibroblasts is Sufficient to Drive Functional Vascularization in Engineered Tissues.

Formation of capillary blood vasculature is a critical requirement for native as well as engineered organs and can be induced in vitro by co-culturing endothelial cells with fibroblasts. However, whether these fibroblasts are required only in the initial morphogenesis of endothelial cells or needed throughout is unknown, and the ability to remove these stromal cells after assembly could be useful for clinical translation. In this study, we introduce a technique termed CAMEO (Controlled Apoptosis in Multicellular Tissues for Engineered Organogenesis), whereby fibroblasts are selectively ablated on demand, and utilize it to probe the dispensability of fibroblasts in vascular morphogenesis. The presence of fibroblasts is shown to be necessary only during the first few days of endothelial cell morphogenesis, after which they can be ablated without significantly affecting the structural and functional features of the developed vasculature. Furthermore, we demonstrate the use of CAMEO to vascularize a construct containing primary human hepatocytes that improved tissue function. In conclusion, this study suggests that transient, initial support from fibroblasts is sufficient to drive vascular morphogenesis in engineered tissues, and this strategy of engineering-via-elimination may provide a new general approach for achieving desired functions and cell compositions in engineered organs.

Somatic Gain of KRAS Function in the Endothelium Is Sufficient to Cause Vascular Malformations That Require MEK but Not PI3K Signaling.

We previously identified somatic activating mutations in the KRAS (Kirsten rat sarcoma viral oncogene homologue) gene in the endothelium of the majority of human sporadic brain arteriovenous malformations; a disorder characterized by direct connections between arteries and veins. However, whether this genetic abnormality alone is sufficient for lesion formation, as well as how active KRAS signaling contributes to arteriovenous malformations, remains unknown. To establish the first in vivo models of somatic KRAS gain of function in the endothelium in both mice and zebrafish to directly observe the phenotypic consequences of constitutive KRAS activity at a cellular level in vivo, and to test potential therapeutic interventions for arteriovenous malformations. Using both postnatal and adult mice, as well as embryonic zebrafish, we demonstrate that endothelial-specific gain of function mutations in Kras (G12D or G12V) are sufficient to induce brain arteriovenous malformations. Active KRAS signaling leads to altered endothelial cell morphogenesis and increased cell size, ectopic sprouting, expanded vessel lumen diameter, and direct connections between arteries and veins. Furthermore, we show that these lesions are not associated with altered endothelial growth dynamics or a lack of proper arteriovenous identity but instead seem to feature exuberant angiogenic signaling. Finally, we demonstrate that KRAS-dependent arteriovenous malformations in zebrafish are refractory to inhibition of the downstream effector PI3K but instead require active MEK (mitogen-activated protein kinase kinase 1) signaling. We demonstrate that active KRAS expression in the endothelium is sufficient for brain arteriovenous malformations, even in the setting of uninjured adult vasculature. Furthermore, the finding that KRAS-dependent lesions are reversible in zebrafish suggests that MEK inhibition may represent a promising therapeutic treatment for arteriovenous malformation patients. Graphical Abstract: A graphical abstract is available for this article.

Inhibition of lysine acetyltransferases impairs tumor angiogenesis acting on both endothelial and tumor cells

BackgroundUnderstanding the signalling pathways involved in angiogenesis, and developing anti-angiogenic drugs are one of the major focuses on cancer research. Herein, we assessed the effect of CPTH6, a lysine acetyltransferase inhibitor and anti-tumoral compound, on angiogenesis-related properties of both endothelial and cancer cells.MethodsThe in vitro effect of CPTH6 on protein acetylation and anti-angiogenic properties on endothelial and lung cancer cells was evaluated via wound healing, trans-well invasion and migration, tube formation, immunoblotting and immunofluorescence. Matrigel plug assay, zebrafish embryo and mouse xenograft models were used to evaluate in vivo anti-angiogenic effect of CPTH6.ResultsCPTH6 impaired in vitro endothelial angiogenesis-related functions, and decreased the in vivo vascularization both in mice xenografts and zebrafish embryos. Mechanistically, CPTH6 reduced α-tubulin acetylation and induced accumulation of acetylated microtubules in the perinuclear region of endothelial cells. Interestingly, CPTH6 also affected the angiogenesis-related properties of lung cancer cells, and conditioned media derived from CPTH6-treated lung cancer cells impaired endothelial cells morphogenesis. CPTH6 also modulated the VEGF/VEGFR2 pathway, and reshaped cytoskeletal organization of lung cancer cells. Finally, anti-migratory effect of CPTH6, dependent on α-tubulin acetylation, was also demonstrated by genetic approaches in lung cancer cells.ConclusionOverall, this study indicates that α-tubulin acetylation could play a role in the anti-angiogenic effect of CPTH6 and, more in general, it adds information to the role of histone acetyltransferases in tumor angiogenesis, and proposes the inhibition of these enzymes as an antiangiogenic therapy of cancer.

Tuning the Local Availability of VEGF within Glycosaminoglycan‐Based Hydrogels to Modulate Vascular Endothelial Cell Morphogenesis

AbstractIncorporation of sulfated glycosaminoglycans (GAGs) into cell‐instructive polymer networks is shown to be instrumental in controlling the diffusivity and activity of growth factors. However, a subtle balance between local retention and release of the factors is needed to effectively direct cell fate decisions. To quantitatively unravel material characteristics governing these key features, the GAG content and the GAG sulfation pattern of star‐shaped poly(ethylene glycol) (starPEG)–GAG hydrogels are herein tuned to control the local availability and bioactivity of GAG‐affine vascular endothelial growth factor (VEGF165). Hydrogels containing varying concentrations of heparin or heparin derivatives with different sulfation pattern are prepared and thoroughly characterized for swelling, mechanical properties, and growth factor transport. Mathematical models are developed to predict the local concentration and spatial distribution of free and bound VEGF165 within the gel matrices. The results of simulation and experimental studies concordantly reveal how the GAG concentration and sulfation pattern determine the local availability of VEGF165 within the cell‐instructive hydrogels and how the factor—in interplay with cell‐instructive gel properties—determines the formation and spatial organization of capillary networks of embedded human vascular endothelial cells. Taken together, this study exemplifies how mathematical modeling and rational hydrogel design can be combined to pave the way for precision tissue engineering.

Dickkopf 2-Expressing Adenovirus Increases the Survival of Random-Pattern Flaps and Promotes Vasculogenesis in a Rat Model.

Dickkopf 2 (DKK2) has important roles in vertebrate development; it inhibits Wnt signaling-related processes, such as axial patterning, limb development, somitogenesis, and eye formation. However, DKK2 also acts as a Wnt signaling agonist. Dickkopf 2, induced during endothelial cell morphogenesis, promotes angiogenesis in cultured human endothelial cells. In this study, we explored the effect of DKK2-expressing adenovirus on random-pattern flaps using a rodent model. A DKK2-expressing (dE1-RGD/DKK2) adenovirus was generated and 20 Sprague-Dawley rats were randomly divided into 2 groups: a DKK2 group and a control group. Each group was intradermally injected with 1 × 10 plaque-forming units of DKK2-expressing adenovirus (DKK2 group) or control virus (control group) 48 hours before and immediately before surgery. Then, random-pattern dorsal cutaneous flaps of 3 × 9 cm were elevated. Flap survival rates and cutaneous blood flow were measured over time, and immunohistochemical staining was performed 10 days after surgery to detect CD31 and vascular endothelial growth factor (VEGF). Immunofluorescence staining confirmed the expression of DKK2 in the DKK2 group. The flap survival rate was higher in the DKK2 group (80.0 ± 4.49%) than in the control group (57.5 ± 4.21%; P < 0.05). Blood flow to the most distal compartment was higher in the DKK2 group than the control group during the early postoperative period. Although vascular density was greater in the DKK2 group, there was no difference in the VEGF concentration between groups. The findings of the present study suggest that the DKK2-expressing adenovirus increases the survival of the random-pattern cutaneous flap independently of VEGF. The administration of the DKK2-expressing adenovirus into elevated skin flaps increased the number of capillaries and blood flow, thereby improving skin flap survival.

Stimulation of 5‐HT1F Receptor Increases Vascular Recovery Following Ischemia/Reperfusion‐Induced AKI

Acute kidney injury (AKI) is a disease with no treatment. After AKI, there is a marked reduction in renal vasculature. Thus, promoting vascular recovery following AKI could facilitate renal repair as the vasculature is responsible for carrying oxygen and nutrients to extravascular tissues. Our laboratory has shown that stimulating mitochondrial biogenesis (MB) through the 5‐HT1F receptor stimulates recovery from AKI and 5‐HT1F receptor knockout mice have decreased MB and poor renal recovery. Importantly, we have shown that stimulation of the 5‐HT1F receptor with LY344864 or lasmiditan induces MB, branching morphogenesis, and migration in human renal glomerular endothelial cells (EC) in vitro. We hypothesize that the 5‐HT1F receptor plays a role in renal vascular recovery following AKI. 5‐HT1F receptor KO mice have decreased CD31+ and aSMA+ ECs in the kidney, suggesting that the 5‐HT1F receptor plays a role in renal vascular homeostasis. To assess the role of the 5‐HT1F receptor in renal vascular injury, WT and 5‐HT1F receptor KO mice were subjected to IR‐induced AKI. 5‐HT1F receptor KO mice exhibited increased renal Evans blue dye (EBD) extravasation in comparison to I/R injured WT mice 24h following injury, suggesting increased vascular injury. To assess the role of 5‐HT1F receptor in renal vascular recovery following IR‐induced AKI, WT mice were treated with LY344864 (2 mg/kg) beginning 24h after I/R‐induced AKI. LY344864 decreased renal EBD extravasation 144h following injury compared to vehicle treated AKI mice, suggesting that stimulation of 5‐HT1F receptor increases vascular recovery. We also assessed whether or not lasmiditan, a more specific and potent 5‐HT1F receptor agonist which has recently been FDA approved, would induce MB in vivo. Mice were treated with lasmiditan (0.3 mg/kg) for 24h had increased protein levels of PGC1a and TFAM, both of which are regulators of MB and markers of MB. Taken together, these data indicate a role for 5‐HT1F receptor signaling in renal vascular injury and repair following I/R‐induced AKI. We propose that inducing MB in ECs after AKI restores vascular function and stimulates renal repair and recovery, and that repurposing lasmiditan as a treatment option for AKI may prove useful.Support or Funding InformationThis work was supported by F32 DK120222‐01A1 to TVD (National Institute of Health) and 1BX000851 (Department of Veterans Affairs) to RGS.

Cholesterol-lowering drug pitavastatin targets lung cancer and angiogenesis via suppressing prenylation-dependent Ras/Raf/MEK and PI3K/Akt/mTOR signaling.

Therapeutic agents that target both tumor cell and vascular endothelial cell may achieve additional anti-tumor efficacy, particularly in lung cancer due to the critical roles of angiogenesis during lung cancer progression and metastasis. In this work, we showed that pitavastatin, a novel cholesterol-lowering drug, potently inhibited lung cancer cells and angiogenesis. This was achieved by the induction of apoptosis and inhibition of proliferation of lung cancer cells and human lung tumor-associated endothelial cell. Pitavastatin was not only effective to chemo-sensitive but also chemo-resistant lung cancer cells. This was also consistent with the finding that pitavastatin significantly enhanced cisplatin's efficacy in lung cancer xenograft model without causing toxicity in mice. We further showed that pitavastatin inhibited lung tumor angiogenesis in vitro and in vivo through suppressing human lung tumor-associated endothelial cell migration and morphogenesis without affecting adhesion. Mechanistically, we showed that pitavastatin acted on lung cancer cells and human lung tumor-associated endothelial cell through suppressing prenylation-dependent Ras/Raf/MEK and PI3K/Akt/mTOR signaling. Our work is the first to demonstrate the inhibitory effects of pitavastatin on Ras-mediated signaling. Our findings provide pre-clinical evidence to repurpose pitavastatin for the treatment of lung cancer.

Harnessing mesenchymal stem cell secretome: Effect of extracellular matrices on proangiogenic signaling.

The low engraftment and retention rate of mesenchymal stem cells (MSCs) at the target site indicates that the potential benefits of MSC-based therapies can be attributed to their paracrine signaling. In this study, the extracellular matrices (ECMs) deposited by bone marrow-derived human MSCs in the presence and absence of ascorbic acid was characterized. MSCs were seeded on top of decellularized ECM (dECM) and the concentrations of proangiogenic and antiangiogenic molecules released in culture (conditioned) media was compared. Effects of ECM derived from MSCs with different passage numbers on MSC secretome was also investigated. Our study revealed that the expression of proangiogenesis-related factors were upregulated when MSCs were harvested on dECMs, irrespective of media supplementation, as compared with those cultured on tissue culture plates. In addition, dECM generated in the presence of ascorbic acid promoted the expression of proangiogenic molecules as compared with dECM-derived in absence of media supplementation. Further, it was observed that the effectiveness of dECM to stimulate proangiogenic signaling of MSCs was reduced as cell passage number was increased from P3 to P5. The proliferation as well as capillary morphogenesis of human umbilical vein endothelial cells (HUVECs) in the presence of conditioned media were enhanced compared with the normal HUVECs culture media. These data indicate that the secretory signatures of MSCs and consequently, the therapeutic efficacy of MSCs can be regulated by presentation of dECM composition and variation of its composition.

Peptidomics analysis of umbilical cord blood reveals potential preclinical biomarkers for neonatal respiratory distress syndrome

AimsThe purpose of this study was to investigate the pathophysiology and discover novel predictors of neonatal respiratory distress syndrome (NRDS) from a peptidomics perspective. Main methodsComparative profiling of umbilical cord blood from NRDS and control patients was performed by liquid chromatography tandem mass spectrometry technology. The underlying biological functions of the differentially expressed peptides (DEPs) were predicted by Gene Ontology (GO) and KEGG pathway analyses. The interactions of DEPs and their precursor proteins were explored by ingenuity pathway analysis (IPA). The sources and stability of DEPs were determined by online databases, including UniProt, SMART and ProtParam tool. Key findingsA total of 251 DEPs were identified, of which 139 peptides were upregulated, and 112 peptides were downregulated (fold change ≥2.0, P < 0.05). These DEPs were predicted to be associated with respiratory failure, atelectasis, and morphogenesis of endothelial cells. These processes indicated that DEPs may play a role in NRDS. Among them, eleven stable DEPs might be used as preclinical biomarkers. SignificanceOur findings improve our understanding of NRDS and facilitate the discovery of candidate diagnostic biomarkers for NRDS from the perspective of peptidomics.

Investigation of Integrated Effects of Hydroxyapatite and VEGF on Capillary Morphogenesis of Endothelial Cells.

Major drawbacks of using autografts and allografts for repairing large bone defects/injuries are the shortage of available tissues and potential disease transmission. Bone tissue engineering is believed to be a promising alternative to bone grafts. The concomitant formation of vascular networks within the tissue engineered constructs for successful bone regeneration still remains a challenge. The physicochemical properties of a tissue microenvironment are known to regulate vascular network formation. However, our understanding of the mechanism, by which endothelial cells integrate these physicochemical signals emanating from the microenvironment to undergo capillary morphogenesis, is limited. In this study, the integrated effects of the vascular endothelial growth factor (VEGF), pro-angiogenic molecule, and hydroxyapatite (HA), widely used as an additive and a scaffold in bone reconstructive surgery, on capillary formation of human umbilical vein endothelial cells (HUVECs) were investigated. Toward this, HUVECs were seeded on scaffolds composed of 15% (w/v) poly(ethylene glycol) diacrylate, 5% (w/v) gelatin methacrylate (GelMA), and 1% (w/v) photoinitiator. The concentration of HA was varied from 0% to 0.2% (w/v). The study demonstrated that HA elicited an inhibitory response. The presence of VEGF promoted a sprouting response with a significant enhancement in the number of sprouts observed in the case of gels with higher concentrations of HA.

Engineering microenvironments towards harnessing pro-angiogenic potential of mesenchymal stem cells

Mesenchymal stem cell (MSC)-based therapy for promoting vascular regeneration is a promising strategy for treating ischemic diseases. However, low engraftment and retention rate of MSCs at the target site highlights the importance of paracrine signaling of MSCs in the reparative process. Thus, harnessing MSC-secretome is essential for rational design of MSC-based therapies. The role of microenvironment in regulating the paracrine signaling of MSCs is not well known. In this study, human bone marrow-derived MSCs were seeded on matrices with varying stiffness or cell adhesive sites, and conditioned media was collected. The concentrations of angiogenic molecules in the media was measured via ELISA. In addition, the bioactivity of the released molecules was investigated via assessing the proliferation and capillary morphogenesis of human umbilical vein endothelial cells (HUVECs) incubated with conditioned media. Our study revealed that secretion of vascular endothelial growth factor (VEGF) is dependent on substrate stiffness. Maximal secretion was observed when MSCs were seeded on hydrogel matrices of 5.0 kPa stiffness. Proliferation and tubulogenesis of HUVECs supported ELISA data. On the other hand, variation of cell adhesive sites while maintaining a uniform optimal stiffness, did not influence the pro-angiogenic activity of MSCs.

YAP and TAZ Negatively Regulate Prox1 During Developmental and Pathologic Lymphangiogenesis.

The Hippo pathway governs cellular differentiation, morphogenesis, and homeostasis, but how it regulates these processes in lymphatic vessels is unknown. We aimed to reveal the role of the final effectors of the Hippo pathway, YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif), in lymphatic endothelial cell (LEC) differentiation, morphogenesis, and homeostasis. During mouse embryonic development, LEC-specific depletion of Yap/Taz disturbed both plexus patterning and valve initiation with upregulated Prox1 (prospero homeobox 1). Conversely, LEC-specific YAP/TAZ hyperactivation impaired lymphatic specification and restricted lymphatic sprouting with profoundly downregulated Prox1. Notably, lymphatic YAP/TAZ depletion or hyperactivation aggravated or attenuated pathological lymphangiogenesis in mouse cornea. Mechanistically, VEGF (vascular endothelial growth factor)-C activated canonical Hippo signaling pathway in LECs. Indeed, repression of PROX1 transcription by YAP/TAZ hyperactivation was mediated by recruitment of NuRD (nucleosome remodeling and histone deacetylase) complex and endogenous binding activity of TEAD (TEA domain family members) to the PROX1 promoter. Furthermore, YAP/TAZ hyperactivation enhanced MYC signaling and inhibited CDKN1C, leading to cell cycle dysregulation and aberrant proliferation. We find that YAP and TAZ play promoting roles in remodeling lymphatic plexus patterning and postnatal lymphatic valve maintenance by negatively regulating Prox1 expression. We further show that YAP and TAZ act as plastic regulators of lymphatic identity and define the Hippo signaling-mediated PROX1 transcriptional programing as a novel dynamic checkpoint underlying LEC plasticity and pathophysiology.

Oleuropein aglycone attenuates the pro-angiogenic phenotype of senescent fibroblasts: A functional study in endothelial cells

The “senescence-associated secretory phenotype” (SASP) supports a pro-tumoral and pro-angiogenic microenvironment through increased secretion of inflammatory and growth factors. We investigated oleuropein aglycone (OLE, 10 µM, 5 weeks) effect on capillary morphogenesis induced by MRC5 fibroblast SASP in mature (hMVEC) and progenitor (ECFC) endothelial cells. In senescent fibroblasts OLE reduced NFkB signaling and the expression of several SASP factors. The levels of IL-8 and VEGF were decreased in senescent fibroblast-conditioned medium collected after OLE treatment (CM senOLE) compared to that from untreated fibroblasts (CM sen). Pre-incubation with CMsen enhanced invasive activity and capillary morphogenesis in endothelial cells. This angiogenic phenotype was significantly attenuated upon pre-incubation with CM senOLE, along with reduced MMP-2, MMP-9 and uPA secretion by endothelial cells. In conclusion, OLE can modulate angiogenesis indirectly acting on local senescent fibroblasts, a new mechanism possibly contributing to the beneficial effects of the Mediterranean diet against cancer and cardiovascular diseases.

Ets-1 drives breast cancer cell angiogenic potential and interactions between breast cancer and endothelial cells.

Ets-1 transcription factor overexpression in breast cancers is associated with invasive features and is associated with a poor prognosis. Beyond its role in driving carcinoma cell invasion, in this study, we wished to determine whether Ets-1 overexpression in cancer cells promotes angiogenesis by creating a paracrine pro-invasive environment for endothelial cells as well. To address this question, we set up different co-culture models of cancer cells with endothelial cells. Conditioned media from cancer cells induced endothelial cell proliferation, migration and morphogenesis in matrix models. Of note, co-culture assays in three-dimensional matrix models also revealed the reciprocal induction of cancer cell morphogenesis by endothelial cells, in support of an angiocrine action on tumor cells. Ets-1 emerged as a key regulator of the angiogenic potential of breast cancer cells, favoring their ability to induce, in a paracrine manner, the morphogenesis of endothelial cells and also to physically interact with the latter. Nevertheless, Ets-1 overexpression in cancer cells also restrained their chemoattractive potential for endothelial cells both in Boyden chambers and in ex vivo 3D co-cultures. Finally, Ets-1 modulation in breast cancer cells qualitatively altered the angiogenic pattern of experimental in vivo tumors, with a balance between vessel recruitment and intratumoral small capillaries sprouting. Taken together, our data highlight a critical and intriguing role for Ets-1 in the angiogenic potential of breast cancer cells, and reveal another facet of Ets-1 oncogenic activities.

Blood concentrations of complement split product iC3b and serum C3 associate with SLE disease activity

The “senescence-associated secretory phenotype” (SASP) supports a pro-tumoral and pro-angiogenic microenvironment through increased secretion of inflammatory and growth factors. We investigated oleuropein aglycone (OLE, 10 µM, 5 weeks) effect on capillary morphogenesis induced by MRC5 fibroblast SASP in mature (hMVEC) and progenitor (ECFC) endothelial cells. In senescent fibroblasts OLE reduced NFkB signaling and the expression of several SASP factors. The levels of IL-8 and VEGF were decreased in senescent fibroblast-conditioned medium collected after OLE treatment (CM senOLE) compared to that from untreated fibroblasts (CM sen). Pre-incubation with CMsen enhanced invasive activity and capillary morphogenesis in endothelial cells. This angiogenic phenotype was significantly attenuated upon pre-incubation with CM senOLE, along with reduced MMP-2, MMP-9 and uPA secretion by endothelial cells. In conclusion, OLE can modulate angiogenesis indirectly acting on local senescent fibroblasts, a new mechanism possibly contributing to the beneficial effects of the Mediterranean diet against cancer and cardiovascular diseases.

Soluble Mediators Produced by Pro-Resolving Macrophages Inhibit Angiogenesis.

Different subtypes of macrophages have been shown to participate in different stages of inflammation and tissue repair. In the late stage of tissue repair, the macrophages, following their engulfment of apoptotic neutrophils, acquire a new phenotype termed alternatively activated macrophages. These macrophages produce growth factors, such as vascular endothelial growth factor (VEGF), that facilitate the angiogenic response as part of tissue restoration. Then, in the later stages of tissue healing, capillary regression takes place. It is presently unknown whether macrophages play an antiangiogenic role in the final stages of tissue repair. Here, we examined whether soluble mediators secreted by pro-resolving CD11blow macrophages (Mres) inhibit angiogenesis in the context of the resolution of tissue repair. Our findings indicate that soluble mediators produced by ex vivo generated Mres (CM-Mres) attenuate angiogenesis in vitro by inhibiting human umbilical vein endothelial cell (HUVEC) proliferation by lowering their cyclin D1 expression. In addition, CM-Mres lowered HUVEC survival by inducing caspase 3/7 activation, and also inhibited VEGFR2 activation via VEGF. HUVEC migration and differentiation to tubular-like structure was also inhibited by CM-Mres. Similarly, CM-Mres significantly inhibited neovascularization as depicted ex vivo by utilizing the rat aorta ring assay and in vivo by utilizing the chick chorioallantoic membrane assay. Notably endostatin, which was shown previously to exert its antiangiogenic effect by inhibiting proliferation, survival, motility, and morphogenesis of endothelial cells via inhibition of VEGFR2 activation, is produced by Mres. Taken together, our results suggest that a specialized subset of macrophages that appear during the resolution of inflammation can produce antiangiogenic mediators, such as endostatin. These mediators can halt angiogenesis, thereby restoring tissue structure.

The dipeptidyl peptidase-4 (DPP-4) inhibitor sitagliptin ameliorates retinal endothelial cell dysfunction triggered by inflammation

Diabetic retinopathy is considered a low-grade chronic inflammatory disease and several inflammatory molecules, including tumor necrosis factor (TNF)-α, are known to play a major role in the degeneration of retinal capillaries. Previous studies have reported that sitagliptin, a DPP-4 inhibitor, prevents the increase in blood-retinal barrier (BRB) permeability and inhibits the tight junction disassembly induced by diabetes. AimOur goal was to investigate whether sitagliptin is able to prevent retinal endothelial cells (EC) dysfunction triggered by the pro-inflammatory cytokine TNF-α. Main methodsThe effects of TNF-α and/or sitagliptin on primary cultures of bovine retinal EC were tested. The EC monolayer permeability was analyzed by using 70 kDa rhodamine isothiocyanate (RITC) dextran. The cellular distribution profile of claudin-5 was examined by immunofluorescence staining, and DPP-4 activity was evaluated by using a fluorogenic substrate. Cell viability was assessed by MTT assay, and cell proliferation by the BrdU incorporation assay. Retinal EC migration and angiogenesis were evaluated by a scratch assay and a capillary tube formation in matrigel assay, respectively. Key findingsTNF-α increased the permeability of EC monolayer and induced the loss of claudin-5 immunostaining at the cell borders. This impairment was associated with decreased migration and capillary morphogenesis of retinal EC. Sitagliptin was unable to prevent the effect of TNF-α on EC permeability. However, it decreased DPP-4 activity in bovine retinal EC exposed to TNF-α, without affecting cell viability. Moreover, sitagliptin enhanced the migration and capillary morphogenesis in bovine retinal EC challenged with TNF-α. SignificanceThese results suggest that sitagliptin is able to positively modulate vascular EC function under conditions of retinal inflammation.