Pro-angiogenic Response Research Articles

A-Kinase Anchoring Protein 2 Promotes Protection against Myocardial Infarction.

Myocardial infarction (MI) is a leading cause of maladaptive cardiac remodeling and heart failure. In the damaged heart, loss of function is mainly due to cardiomyocyte death and remodeling of the cardiac tissue. The current study shows that A-kinase anchoring protein 2 (AKAP2) orchestrates cellular processes favoring cardioprotection in infarcted hearts. Induction of AKAP2 knockout (KO) in cardiomyocytes of adult mice increases infarct size and exacerbates cardiac dysfunction after MI, as visualized by increased left ventricular dilation and reduced fractional shortening and ejection fraction. In cardiomyocytes, AKAP2 forms a signaling complex with PKA and the steroid receptor co-activator 3 (Src3). Upon activation of cAMP signaling, the AKAP2/PKA/Src3 complex favors PKA-mediated phosphorylation and activation of estrogen receptor α (ERα). This results in the upregulation of ER-dependent genes involved in protection against apoptosis and angiogenesis, including Bcl2 and the vascular endothelial growth factor a (VEGFa). In line with these findings, cardiomyocyte-specific AKAP2 KO reduces Bcl2 and VEGFa expression, increases myocardial apoptosis and impairs the formation of new blood vessels in infarcted hearts. Collectively, our findings suggest that AKAP2 organizes a transcriptional complex that mediates pro-angiogenic and anti-apoptotic responses that protect infarcted hearts.

Peritumoral B cells drive proangiogenic responses in HMGB1-enriched esophageal squamous cell carcinoma

Several B-cell subsets with distinct functions and polarized cytokine profiles that extend beyond antibody production have been reported in different cancers. Here we have demonstrated that proliferating B cells were predominantly found in the peritumoral region of esophageal squamous cell carcinoma (ESCC). These B cells were enriched in tumor nests with high expression of high-mobility group box 1 (HMGB1). High densities of peritumoral proliferating B cells and concomitantly high intratumoral HMGB1 expression showed improved prognostic significance, surpassing prognostic stratification of ESCC patients based on HMGB1 positivity alone. This striking association led us to set up models to test whether cancer-derived HMGB1 could shape tumor microenvironment via modulation on B cells. Overexpression of HMGB1 in ESCC cell lines (KYSE510 and EC18) enhanced proliferation and migration of B cells. Transcriptomic analysis showed that migratory B cells exhibited high enrichment of proangiogenic genes. VEGF expression in proliferating B cells was induced upon co-culture of HMGB1-overexpressing tumor cells and B cells. Secretome array profiling of conditioned media (CM) from the co-culture revealed rich expression of proangiogenic proteins. Consequently, incubation of human umbilical vein endothelial cells with CM promoted angiogenesis in tube formation and migration assays. HMGB1 inhibitor, glycyrrhizin, abolishes all the observed proangiogenic phenotypes. Finally, co-injection of B cells and CM with HMGB1-overexpressing tumor cells, but not with glycyrrhizin, significantly enhanced tumor growth associated with increased microvascular density in ESCC xenograft mice model. Our results indicate that cancer-derived HMGB1 elevates angiogenesis in ESCC by shifting the balance toward proangiogenic signals in proliferating B cells.

Three-dimensional microengineered vascularised endometrium-on-a-chip.

STUDY QUESTIONCan we reconstitute physiologically relevant 3-dimensional (3D) microengineered endometrium in-vitro model?SUMMARY ANSWEROur representative microengineered vascularised endometrium on-a-chip closely recapitulates the endometrial microenvironment that consists of three distinct layers including epithelial cells, stromal fibroblasts and endothelial cells in a 3D extracellular matrix in a spatiotemporal manner.WHAT IS KNOWN ALREADYOrgan-on-a-chip, a multi-channel 3D microfluidic cell culture system, is widely used to investigate physiologically relevant responses of organ systems.STUDY DESIGN, SIZE, DURATIONThe device consists of five microchannels that are arrayed in parallel and partitioned by array of micropost. Two central channels are for 3D culture and morphogenesis of stromal fibroblast and endothelial cells. In addition, the outermost channel is for the culture of additional endometrial stromal fibroblasts that secrete biochemical cues to induce directional pro-angiogenic responses of endothelial cells. To seed endometrial epithelial cells, on Day 8, Ishikawa cells were introduced to one of the two medium channels to adhere on the gel surface. After that, the microengineered endometrium was cultured for an additional 5–6 days (total ∼ 14 days) for the purpose of each experiment.PARTICIPANTS/MATERIALS, SETTING, METHODSMicrofluidic 3D cultures were maintained in endothelial growth Medium 2 with or without oestradiol and progesterone. Some cultures additionally received exogenous pro-angiogenic factors. For the three distinct layers of microengineered endometrium-on-a-chip, the epithelium, stroma and blood vessel characteristics and drug response of each distinct layer in the microfluidic model were assessed morphologically and biochemically. The quantitative measurement of endometrial drug delivery was evaluated by the permeability coefficients.MAIN RESULTS AND THE ROLE OF CHANCEWe established microengineered vascularised endometrium-on-chip, which consists of three distinct layers: epithelium, stroma and blood vessels. Our endometrium model faithfully recapitulates in-vivo endometrial vasculo-angiogenesis and hormonal responses displaying key features of the proliferative and secretory phases of the menstrual cycle. Furthermore, the effect of the emergency contraception drug levonorgestrel was evaluated in our model demonstrating increased endometrial permeability and blood vessel regression in a dose-dependent manner. We finally provided a proof of concept of the multi-layered endometrium model for embryo implantation, which aids a better understanding of the molecular and cellular mechanisms underlying this process.LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONThis report is largely an in-vitro study and it would be beneficial to validate our findings using human primary endometrial cells.WIDER IMPLICATIONS OF THE FINDINGSOur 3D microengineered vascularised endometrium-on-a-chip provides a new in-vitro approach to drug screening and drug discovery by mimicking the complicated behaviours of human endometrium. Thus, we suggest our model as a tool for addressing critical challenges and unsolved problems in female diseases, such as endometriosis, uterine cancer and female infertility, in a personalised manner.STUDY FUNDING/COMPETING INTEREST(S)This work is supported by funding from the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) to Y.J.K. (No. 2018R1C1B6003), to J.A. (No. 2020R1I1A1A01074136) and to H.S.K. (No. 2020R1C1C100787212). The authors report no conflicts of interest.

Interleukin-17A Triggers the Release of Platelet-Derived Factors Driving Vascular Endothelial Cells toward a Pro-Angiogenic State.

Platelets comprise a highly interactive immune cell subset of the circulatory system traditionally known for their unique haemostatic properties. Although platelets are considered as a vault of growth factors, cytokines and chemokines with pivotal role in vascular regeneration and angiogenesis, the exact mechanisms by which they influence vascular endothelial cells (ECs) function remain underappreciated. In the present study, we examined the role of human IL-17A/IL-17RA axis in platelet-mediated pro-angiogenic responses. We reveal that IL-17A receptor (IL-17RA) mRNA is present in platelets transcriptome and a profound increase is documented on the surface of activated platelets. By quantifying the protein levels of several factors, involved in angiogenesis, we identified that IL-17A/IL17RA axis selectively induces the release of vascular endothelial growth factor, interleukin -2 and -4, as well as monocyte chemoattractant protein -1 from treated platelets. However, IL-17A exerted no effect on the release of IL-10, an anti-inflammatory factor with potentially anti-angiogenic properties, from platelets. Treatment of human endothelial cell two-dimensional tubule networks or three-dimensional spheroid and mouse aortic ring structures with IL-17A-induced platelet releasate evoked pro-angiogenic responses of ECs. Our findings suggest that IL-17A may critically affect platelet release of pro-angiogenic factors driving ECs towards a pro-angiogenic state.

Nanoengineered biomimetic Cu-based nanoparticles for multifunational and efficient tumor treatment

The microwave dynamic therapy (MDT) mediated by cytotoxic reactive oxygen species (ROS) is a promising anticancer therapeutic method. However, the therapeutic efficiency of MDT is restricted by several limitations including insufficient ROS generation, strong proangiogenic response, and low tumor-targeting efficiency. Herein, we find that Cu-based nanoparticles can produce oxygen under microwave (MW) irradiation to raise the generation of ROS, such as •O2, •OH and 1O2, especially •O2. On this basis, a nanoengineered biomimetic strategy is designed to improve the efficiency of MDT. After intravenous administration, the nanoparticles accumulate to the tumor site through targeting effect mediated by biomimetic modification, and it can continuously produce oxygen to raise the levels of ROS in tumor microenvironment under MW irradiation for MDT. Additionally, Apatinib is incorporated as antiangiogenic drug to downregulate the expression of vascular endothelial growth factor (VEGF), which can effectively inhibit the tumor angiogenesis after MDT. Hence, the tumor inhibition rate is as high as 96.79%. This study provides emerging strategies to develop multifunctional nanosystems for efficient tumor therapy by MDT.

Exosomes from cervical cancer cells facilitate pro-angiogenic endothelial reconditioning through transfer of Hedgehog\u2013GLI signaling components

BackgroundAngiogenic switch is a hallmark feature of transition from low-grade to high-grade cervical intraepithelial neoplasia (CIN) in cervical cancer progression. Therefore, early events leading to locally-advanced cervical metastatic lesions demand a greater understanding of the underlying mechanisms. Recent leads indicate the role of tumor-derived exosomes in altering the functions of endothelial cells in cervical cancer, which needs further investigation.MethodsExosomes isolated from cervical cancer cell lines were assessed for their angiogenic effect on the human umbilical vein endothelial cells (HUVEC) using tube formation and wound healing assay. The exosomal uptake by HUVEC cells was monitored using PKH-67 labelling followed by fluorescence microscopy. Alterations in Hh-GLI signaling components, PTCH1 and GLI1, in HUVEC were measured by immunoblotting. Changes in angiogenesis-related transcripts of vascular endothelial growth factor VEGF-A, VEGF-B, VEGFR2 and angiopoietin-1, angiopoietin-2, osteopontin were measured in exosome-treated HUVEC and in the exosomal RNA by RT-PCR.ResultsEnhanced tube formation, with an increased number of nodes and branching was observed in HUVEC’s treated with exosomes derived from different cervical cancer cell lines. HPV-positive (SiHa and HeLa) cells’ exosomes were more angiogenic. Exosome-treated HUVEC showed increased migration rate. PKH-67 labelled exosomes were found internalized in HUVEC. A high level of PTCH1 protein was detected in the exosome—treated endothelial cells. Subsequent RT-PCR analysis showed increased transcripts of Hh-GLI downstream target genes VEGF-A, VEGFR2, angiopoietin-2, and decreased expression of VEGF-B, and angiopoietin-1, suggestive of active Hh-GLI signaling. These effects were more pronounced in HUVEC’s treated with exosomes of HPV-positive cells. However, these effects were independent of tumor-derived VEGF-A as exosomal cargo lacked VEGF-A transcripts or proteins.ConclusionOverall, the data showed cervical cancer exosomes promote pro-angiogenic response in endothelial cells via upregulation of Hh-GLI signaling and modulate downstream angiogenesis-related target genes. The study provides a novel exosome-mediated mechanism potentially favoring cervical angiogenesis and thus identifies the exosomes as potential pharmacological targets against locally-advanced metastatic cervical lesions.Graphic abstract

Characterization of p38 Inflammatory Response in Human Brain Vascular Pericyte Cells

G protein-coupled receptor (GPCR) mediated mitogen activated protein kinase (MAPK) p38 activation is a crucial regulator of pro-inflammatory and pro-angiogenic responses. Prior studies have revealed an atypical pathway for p38 activation that induces auto-phosphorylation of p38 through the direct binding of transforming growth factor β activated kinase 1 binding protein-1 (TAB1) in endothelial cells. During angiogenesis and inflammation, endothelial cells are known for their rapid proliferation, migration, and expression of cytokines. Pericyte cells serve to guide these endothelial cells, modulating vascular stability and orchestrating vascular inflammatory responses. During inflammation, pericytes migrate away from the vasculature leaving endothelial tubules exposed and fragile. Atypical p38 activation has not yet been examined in pericyte cells. We hypothesize that atypical p38 signaling regulates G protein-coupled receptor-mediated vascular function through control of pericyte signaling. Our preliminary data shows for the first time that a family of GPCR ligands, including thrombin and histamine, can activate p38 via the atypical p38 signaling pathway in primary human brain vascular pericytes. Chemical inhibition and siRNA depletion of TAB1-TAB2 abrogates inflammatory signaling by brain pericyte cells. As the role of GPCR induced p38 signaling in pericytes has not been examined, our current studies focus on defining this role, including establishing a co-cultured model of pericyte and endothelial cells. Using this model, we are the first to define atypical p38 signaling in pericyte cytokine expression, migration, and endothelial vascular formation. These studies represent an unexplored branch of neurovascular inflammatory signaling, which we predict is a critical driver of neurovascular disease.

Transcriptional Regulation of Thrombin-Induced Endothelial VEGF Induction and Proangiogenic Response

Thrombin, the ligand of the protease-activated receptor 1 (PAR1), is a well-known stimulator of proangiogenic responses in vascular endothelial cells (ECs), which are mediated through the induction of vascular endothelial growth factor (VEGF). However, the transcriptional events underlying this thrombin-induced VEGF induction and angiogenic response are less well understood at present. As reported here, we conducted detailed promotor activation and signal transduction pathway studies in human microvascular ECs, to decipher the transcription factors and the intracellular signaling events underlying the thrombin and PAR-1-induced endothelial VEGF induction. We found that c-FOS is a key transcription factor controlling thrombin-induced EC VEGF synthesis and angiogenesis. Upon the binding and internalization of its G-protein-coupled PAR-1 receptor, thrombin triggers ERK1/2 signaling and activation of the nuclear AP-1/c-FOS transcription factor complex, which then leads to VEGF transcription, extracellular secretion, and concomitant proangiogenic responses of ECs. In conclusion, exposure of human microvascular ECs to thrombin triggers signaling through the PAR-1–ERK1/2–AP-1/c-FOS axis to control VEGF gene transcription and VEGF-induced angiogenesis. These observations offer a greater understanding of endothelial responses to thromboinflammation, which may help to interpret the results of clinical trials tackling the conditions associated with endothelial injury and thrombosis.

Impact of ultraviolet radiation and exposome on rosacea: Key role of photoprotection in optimizing treatment.

BackgroundPathophysiology of rosacea is not completely understood and involves a complex interaction among genetics, ultraviolet (UV) light, microorganisms, impaired skin barrier, neuronal and vascular dysfunction, and immune system disruption.AimsTo describe the etiology of rosacea with an emphasis on the role of UV radiation and exposome, and to review the importance of non‐pharmacologic strategies focusing on photoprotection.MethodsWe conducted a narrative review of the literature. We performed literature searches with PubMed from January 1990 to November 2020 using the keywords “rosacea”, “pathogenesis”, “ultraviolet radiation”, “exposome”, “photoprotection”, “sunscreens” and “non‐pharmacologic agents”. The search was limited to English, Spanish, and French language articles.ResultsSeveral environmental factors such as UV light, diverse microorganisms, air pollution, tobacco smoking, nutrition, and psychological stress showed to trigger or worsen rosacea. UV radiation was reported to induce pro‐inflammatory, pro‐angiogenic, and pro‐fibrotic responses. We found 6 original articles about the impact of sunscreens on rosacea. The use of sunscreens containing ingredients with emollient, anti‐inflammatory, and/or vasoregulatory properties was shown to significantly improve symptomatology.ConclusionUV radiation and the exposome play a key role in the development of rosacea. UV light is implicated in all significant aspects of rosacea: skin inflammation, neoangiogenesis, telangiectasia, and fibrosis, and may even initiate rosacea. While the use of sunscreens is widely recommended, the literature on the impact of photoprotection in rosacea is scarce. Adequately formulated sunscreens could not only provide the required level of photoprotection, but may also help to mitigate the barrier dysfunction, neutralize facial redness (tinted sunscreens), and decrease inflammation and vascular dysfunction.

Assessing the Angiogenic Efficacy of Pleiotrophin Released from Injectable Heparin-Alginate Gels.

With this work, we design alginate-based hydrogels for therapeutically directing revascularization and repair processes in vivo. We immobilize pleiotrophin (PTN) in injectable hydrogel formulations as the target factor to stimulate proangiogenic responses in endothelial cells. The optimized heparin-alginate/chitosan hydrogels, produced by internal crosslinking with calcium carbonate, show good biocompatibility and injectability and allow controlling the release of immobilized proteins in the subcutaneous tissue over a period of 7 days. In vitro assays, performed with translational human induced pluripotent stem cell-derived endothelial cells, and the in vivo Matrigel plug assay are conducted to demonstrate the angiogenic effects of PTN on endothelial cells. Our results indicate that PTN stimulates endothelial cell morphogenesis in vitro and the migration of endothelial cells and macrophages as soon as 4 days after injections of the developed hydrogels, promoting the formation of structures similar to the healthy granulation tissue, which is an indicator of healing in ischemic wounds. These studies provide the rationale for further investigating this novel therapeutic for pursuing increased vascular density for efficient regeneration of ischemic tissues, by leveraging the host endothelial cell population to initiate angiogenic and reparative processes in vivo. Impact statement Localized, sustained, and controlled delivery of angiogenic factors is crucial for enabling the formation of novel vascular networks in ischemic tissues. This study describes the development of an injectable heparin-alginate/collagen hydrogel for controlling the in vivo release and bioactivity of pleiotrophin (PTN), a heparin-binding factor with significant angiogenic activity. We demonstrate that PTN promotes angiogenesis in an in vitro model of hypoxia and in preclinical subcutaneous models. These results advance our understanding of PTN function in guiding therapeutic angiogenesis and are critical to inform the development of novel translational strategies for ischemic tissue repair and regeneration.

Extracellular Vesicle-Derived microRNAs of Human Wharton's Jelly Mesenchymal Stromal Cells May Activate Endogenous VEGF-A to Promote Angiogenesis.

Despite low levels of vascular endothelial growth factor (VEGF)-A, the secretome of human Wharton’s jelly (WJ) mesenchymal stromal cells (MSCs) effectively promoted proangiogenic responses in vitro, which were impaired upon the depletion of small (~140 nm) extracellular vesicles (EVs). The isolated EVs shared the low VEGF-A profile of the secretome and expressed five microRNAs, which were upregulated compared to fetal dermal MSC-derived EVs. These upregulated microRNAs exclusively targeted the VEGF-A gene within 54 Gene Ontology (GO) biological processes, 18 of which are associated with angiogenesis. Moreover, 15 microRNAs of WJ-MSC-derived EVs were highly expressed (Ct value ≤ 26) and exclusively targeted the thrombospondin 1 (THBS1) gene within 75 GO biological processes, 30 of which are associated with the regulation of tissue repair. The relationship between predicted microRNA target genes and WJ-MSC-derived EVs was shown by treating human umbilical-vein endothelial cells (HUVECs) with appropriate doses of EVs. The exposure of HUVECs to EVs for 72 h significantly enhanced the release of VEGF-A and THBS1 protein expression compared to untreated control cells. Finally, WJ-MSC-derived EVs stimulated in vitro tube formation along with the migration and proliferation of HUVECs. Our findings can contribute to a better understanding of the molecular mechanisms underlying the proangiogenic responses induced by human umbilical cord-derived MSCs, suggesting a key regulatory role for microRNAs delivered by EVs.

SDF-1α Gene-Activated Collagen Scaffold Restores Pro-Angiogenic Wound Healing Features in Human Diabetic Adipose-Derived Stem Cells.

Non-healing diabetic foot ulcers (DFUs) can lead to leg amputation in diabetic patients. Autologous stem cell therapy holds some potential to solve this problem; however, diabetic stem cells are relatively dysfunctional and restrictive in their wound healing abilities. This study sought to explore if a novel collagen–chondroitin sulfate (coll–CS) scaffold, functionalized with polyplex nanoparticles carrying the gene encoding for stromal-derived factor-1 alpha (SDF-1α gene-activated scaffold), can enhance the regenerative functionality of human diabetic adipose-derived stem cells (ADSCs). We assessed the impact of the gene-activated scaffold on diabetic ADSCs by comparing their response against healthy ADSCs cultured on a gene-free scaffold over two weeks. Overall, we found that the gene-activated scaffold could restore the pro-angiogenic regenerative response in the human diabetic ADSCs similar to the healthy ADSCs on the gene-free scaffold. Gene and protein expression analysis revealed that the gene-activated scaffold induced the overexpression of SDF-1α in diabetic ADSCs and engaged the receptor CXCR7, causing downstream β-arrestin signaling, as effectively as the transfected healthy ADSCs. The transfected diabetic ADSCs also exhibited pro-wound healing features characterized by active matrix remodeling of the provisional fibronectin matrix and basement membrane protein collagen IV. The gene-activated scaffold also induced a controlled pro-healing response in the healthy ADSCs by disabling early developmental factors signaling while promoting the expression of tissue remodeling components. Conclusively, we show that the SDF-1α gene-activated scaffold can overcome the deficiencies associated with diabetic ADSCs, paving the way for autologous stem cell therapies combined with novel biomaterials to treat DFUs.

Anti-Ageing Protein β-Klotho Rejuvenates Diabetic Stem Cells for Improved Gene-Activated Scaffold Based Wound Healing.

Skin wounds can lead to serious morbidity complications in diabetic patients due to the reduced healing potential of autologous stem cells. One reason for the low functional potency of stem cells from diabetic patients (diabetic stem cells) is attributed to their senescent-like nature. Here, we investigated if an anti-ageing protein, β-klotho, could be used to rejuvenate diabetic stem cells and to promote pro-angiogenic gene-activated scaffold (GAS)-induced functional response for wound healing applications. Human stem cells derived from the adipose tissue (adipose-derived stem cells (ADSCs)) of normal and diabetic (type 2) donors were used for the study. We report that the β-klotho priming facilitated inflammatory signal pruning by reducing interleukin-8 release by more than half while concurrently doubling the release of monocyte chemoattractant protein-1. Additionally, β-klotho priming enhanced the pro-angiogenic response of diabetic ADSCs on GAS by dampening the release of anti-angiogenic factors (i.e., pigment epithelium-derived factor, tissue inhibitor of metalloproteinase-1 and thrombospondin-1) while simultaneously supporting the expression of pro-angiogenic factors (i.e., Vascular Endothelial Growth Factor (VEGF), angiopoietin-2 and angiogenin). Finally, we show that β-klotho pre-treatment expedites the cellular expression of matrix proteins such as collagen IV and collagen VI, which are implicated in tissue maturation. Taken together, our study provides evidence that the synergistic effect of the pro-angiogenic GAS and β-klotho activation effectively accelerates the functional development of diabetic ADSCs for wound healing applications.

Sex Differences in the Pro-Angiogenic Response of Human Endothelial Cells: Focus on PFKFB3 and FAK Activation.

Female hormones and sex-specific factors are established determinants of endothelial function, yet their relative contribution to human endothelium phenotypes has not been defined. Using human umbilical vein endothelial cells (HUVECs) genotyped by donor's sex, we investigated the influence of sex and estrogenic agents on the main steps of the angiogenic process and on key proteins governing HUVEC metabolism and migratory properties. HUVECs from female donors (fHUVECs) showed increased viability (p < 0.01) and growth rate (p < 0.01) compared with those from males (mHUVECs). Despite higher levels of G-protein coupled estrogen receptor (GPER) in fHUVECs (p < 0.001), treatment with 17β-estradiol (E2) and the selective GPER agonist G1 (both 1–100 nM) did not affect HUVEC viability. Migration and tubularization in vitro under physiological conditions were higher in fHUVECs than in mHUVECs (p < 0.05). E2 treatment (1–100 nM) upregulated the glycolytic activator PFKFB3 with higher potency in fHUVECs than in mHUVECs, despite comparable baseline levels. Moreover, Y576/577 phosphorylation of focal adhesion kinase (FAK) was markedly enhanced in fHUVECs (p < 0.001), despite comparable Src activation levels. While the PI3K inhibitor LY294002 (25 µM) inhibited HUVEC migration (p < 0.05), Akt phosphorylation levels in fHUVECs and mHUVECs were comparable. Finally, digitoxin treatment, which inhibits Y576/577 FAK phosphorylation, abolished sexual dimorphism in HUVEC migration. These findings unravel complementary modulation of HUVEC functional phenotypes and signaling molecules involved in angiogenesis by hormone microenvironment and sex-specific factors, and highlight the need for sex-oriented pharmacological targeting of endothelial function.

SDF-1α gene-activated collagen scaffold drives functional differentiation of human Schwann cells for wound healing applications.

Enhancing angiogenesis is the prime target of current biomaterial-based wound healing strategies. However, these approaches largely overlook the angiogenic role of the cells of the nervous system. Therefore, we explored the role of a collagen-chondroitin sulfate scaffold functionalized with a proangiogenic gene stromal-derived factor-1α (SDF-1α)-an SDF-1α gene-activated scaffold on the functional regulation of human Schwann cells (SCs). A preliminary 2D study was conducted by delivering plasmids encoding for the SDF-1α gene into a monolayer of SCs using polyethyleneimine-based nanoparticles. The delivery of the SDF-1α gene into the SCs enhanced the production of proangiogenic vascular endothelial growth factor (VEGF). Subsequently, we investigated the impact of SDF-1α gene-activated scaffold (3D) on the SCs for 2weeks, using a gene-free scaffold as control. The transfection of the SCs within the gene-activated scaffold resulted in transient overexpression of SDF-1α transcripts and triggered the production of bioactive VEGF that enhanced endothelial angiogenesis. The overexpression of SDF-1α also caused transient activation of the transcription factor c-Jun and supported the differentiation of SCs towards a repair phenotype. This was characterized by elevated expression of neurotrophin receptor p75NGFR. During this developmental stage, the SCs also performed an extensive remodelling of the basement matrix (fibronectin, collagen IV, and laminin) to enrich their environment with the pro-neurogenic matrix protein laminin, revealing an enhanced pro-neurogenic behavior. Together, this study shows that SDF-1α gene-activated scaffold is a highly bioinstructive scaffold capable of enhancing proangiogenic regenerative response in human SCs for improved wound healing.

TAMI-10. CIRCULATING ANGIOGENIC CELLS (CACS) IN GLIOBLASTOMA: TOWARDS DEFINING CRUCIAL FUNCTIONAL DIFFERENCES IN CAC-INDUCED NEOPLASTIC VERSUS REACTIVE NEOVASCULARIZATION

Abstract In order to identify suitable therapeutic targets for glioma anti-angiogenic therapy, the process of neovascularization mediated by circulating angiogenic cells (CACs) needs to be scrutinized. In the present study we compared the expression of neovascularization-related genes by three circulating CAC subsets (HPCs, CD34+ and KDR+ cells; internal controls: PBMCs and circulating endothelial cells) of treatment-naïve patients with glioblastoma (GBM) to those of patients undergoing reactive neovascularization (myocardial infarction (MI). CACs from umbilical cord (representing developmental neovascularization) and healthy subjects served as controls. Fluorescent activated cell sorting was used to isolate CACs, RT-PCR to determine the expression levels of a panel of 48 neovascularization-related genes, Luminex assays to measure plasma levels of 21 CAC-related circulating molecules. We found essential differences in gene expression between GBM and MI CACs. GBM CACs had a higher expression of pro-angiogenic factors (esp. KITL, CXCL12 and JAG1); growth factor and chemotactic receptors (IGF1R, TGFbR2, CXCR4 and CCR2); adhesion receptor monomers (ITGA5 and ITGA6) and matricellular factor POSTN. In addition, we found major differences in the levels of neovascularization-related plasma factors. A strong positive correlation between plasma MMP9 levels and expression of CXCR4 in the CAC subset of hematopoietic progenitor cells (HPCs) was found in GBM patients. Our findings indicate that CAC-mediated neovascularization in GBM is characterized by more efficient CAC homing to target tissue and a more potent pro-angiogenic response than in physiologic tissue repair in MI. Our findings can aid in selecting targets for therapeutic strategies acting against GBM-specific CACs.

Metabolic Syndrome Triggered by Fructose Diet Impairs Neuronal Function and Vascular Integrity in ApoE-KO Mouse Retinas: Implications of Autophagy Deficient Activation.

Metabolic syndrome is a disorder characterized by a constellation of clinical findings such as elevated blood glucose, hyperinsulinemia, dyslipidemia, hypertension, and obesity. A positive correlation has been found between metabolic syndrome or its components and retinopathy, mainly at microvascular level, in patients without a history of diabetes. Here, we extend the investigations beyond the vascular component analyzing functional changes as well as neuronal and glial response in retinas of Apolipoprotein E knockout (ApoE-KO) mice fed with 10% w/v fructose diet. Given that autophagy dysfunction is implicated in retinal diseases related to hyperglycemia and dyslipidemia, the activation of this pathway was also analyzed. Two months of fructose intake triggered metabolic derangements in ApoE-KO mice characterized by dyslipidemia, hyperglycemia and hyperinsulinemia. An increased number of TUNEL positive cells, in addition to the ganglion cell layer, was observed in the inner nuclear layer in retina. Vascular permeability, evidenced by albumin–Evans blue leakage and extravasation of albumin was also detected. Furthermore, a significant decrease of the glial fibrillary acidic protein expression was confirmed by Western blot analysis. Absence of both Müller cell gliosis and pro-angiogenic response was also demonstrated. Finally, retinas of ApoE-KO FD mice showed defective autophagy activation as judged by LC3B mRNA and p62 protein levels correlating with the increased cell death. These results demonstrated that FD induced in ApoE-KO mice biochemical alterations compatible with metabolic syndrome associated with neuronal impairment and mild vascular alterations in the retina.

Advantages in Wound Healing Process in Female Mice Require Upregulation A2A-Mediated Angiogenesis under the Stimulation of 17β-Estradiol.

Estrogenic steroids and adenosine A2A receptors promote the wound healing and angiogenesis processes. However, so far, it is unclear whether estrogen may regulate the expression and pro-angiogenic activity of A2A receptors. Using in vivo analyses, we showed that female wild type (WT) mice have a more rapid wound healing process than female or male A2A-deficient mice (A2AKO) mice. We also found that pulmonary endothelial cells (mPEC) isolated from female WT mice showed higher expression of A2A receptor than mPEC from male WT mice. mPEC from female WT mice were more sensitive to A2A-mediated pro-angiogenic response, suggesting an ER and A2A crosstalk, which was confirmed using cells isolated from A2AKO. In those female cells, 17β-estradiol potentiated A2A-mediated cell proliferation, an effect that was inhibited by selective antagonists of estrogen receptors (ER), ERα, and ERβ. Therefore, estrogen regulates the expression and/or pro-angiogenic activity of A2A adenosine receptors, likely involving activation of ERα and ERβ receptors. Sexual dimorphism in wound healing observed in the A2AKO mice process reinforces the functional crosstalk between ER and A2A receptors.

Role of IL-4 in bone marrow driven dysregulated angiogenesis and age-related macular degeneration.

Age-associated sterile inflammation can cause dysregulated choroidal neovascularization (CNV) as age-related macular degeneration (AMD). Intraocular fluid screening of 234 AMD patients identified high levels of IL-4. The purpose of this study was to determine the functional role of IL-4 in CNV formation using murine CNV model. Our results indicate that the IL-4/IL-4 receptors (IL4Rs) controlled tube formation and global proangiogenic responses of bone marrow cells. CCR2+ bone marrow cells were recruited to form very early CNV lesions. IL-4 rapidly induces CCL2, which enhances recruitment of CCR2+ bone marrow cells. This in vivo communication, like quorum-sensing, was followed by the induction of IL-4 by the bone marrow cells during the formation of mature CNVs. For CNV development, IL-4 in bone marrow cells are critically required, and IL-4 directly promotes CNV formation mainly by IL-4R. The IL-4/IL-4Rα axis contributes to pathological angiogenesis through communications with bone marrow cells leading to retinal degeneration.

Hypergravity Activates a Pro-Angiogenic Homeostatic Response by Human Capillary Endothelial Cells.

Capillary endothelial cells are responsible for homeostatic responses to organismic and environmental stimulations. When malfunctioning, they may cause disease. Exposure to microgravity is known to have negative effects on astronauts’ physiology, the endothelium being a particularly sensitive organ. Microgravity-related dysfunctions are striking similar to the consequences of sedentary life, bed rest, and ageing on Earth. Among different countermeasures implemented to minimize the effects of microgravity, a promising one is artificial gravity. We examined the effects of hypergravity on human microvascular endothelial cells of dermal capillary origin (HMEC-1) treated at 4 g for 15 min, and at 20 g for 15 min, 3 and 6 h. We evaluated cell morphology, gene expression and 2D motility and function. We found a profound rearrangement of the cytoskeleton network, dose-dependent increase of Focal Adhesion kinase (FAK) phosphorylation and Yes-associated protein 1 (YAP1) expression, suggesting cell stiffening and increased proneness to motility. Transcriptome analysis showed expression changes of genes associated with cardiovascular homeostasis, nitric oxide production, angiogenesis, and inflammation. Hypergravity-treated cells also showed significantly improved motility and function (2D migration and tube formation). These results, expanding our knowledge about the homeostatic response of capillary endothelial cells, show that adaptation to hypergravity has opposite effect compared to microgravity on the same cell type.