Pro-angiogenic Genes Research Articles

Pre-Conditioning with IFN-γ and Hypoxia Enhances the Angiogenic Potential of iPSC-Derived MSC Secretome.

Induced pluripotent stem cell (iPSC) derived mesenchymal stem cells (iMSCs) represent a promising source of progenitor cells for approaches in the field of bone regeneration. Bone formation is a multi-step process in which osteogenesis and angiogenesis are both involved. Many reports show that the secretome of mesenchymal stromal stem cells (MSCs) influences the microenvironment upon injury, promoting cytoprotection, angiogenesis, and tissue repair of the damaged area. However, the effects of iPSC-derived MSCs secretome on angiogenesis have seldom been investigated. In the present study, the angiogenic properties of IFN-γ pre-conditioned iMSC secretomes were analyzed. We detected a higher expression of the pro-angiogenic genes and proteins of iMSCs and their secretome under IFN-γ and hypoxic stimulation (IFN-H). Tube formation and wound healing assays revealed a higher angiogenic potential of HUVECs in the presence of IFN-γ conditioned iMSC secretome. Sprouting assays demonstrated that within Coll/HA scaffolds, HUVECs spheroids formed significantly more and longer sprouts in the presence of IFN-γ conditioned iMSC secretome. Through gene expression analyses, pro-angiogenic genes (FLT-1, KDR, MET, TIMP-1, HIF-1α, IL-8, and VCAM-1) in HUVECs showed a significant up-regulation and down-regulation of two anti-angiogenic genes (TIMP-4 and IGFBP-1) compared to the data obtained in the other groups. Our results demonstrate that the iMSC secretome, pre-conditioned under inflammatory and hypoxic conditions, induced the highest angiogenic properties of HUVECs. We conclude that pre-activated iMSCs enhance their efficacy and represent a suitable cell source for collagen/hydroxyapatite with angiogenic properties.

Endothelial Phospholipase Cγ2 Improves Outcomes of Diabetic Ischemic Limb Rescue Following VEGF Therapy.

Therapeutic vascular endothelial growth factor (VEGF) replenishment has met with limited success for the management of critical limb-threatening ischemia. To improve outcomes of VEGF therapy, we applied single-cell RNA sequencing (scRNA-seq) technology to study the endothelial cells of the human diabetic skin. Single-cell suspensions were generated from the human skin followed by cDNA preparation using the Chromium Next GEM Single-cell 3' Kit v3.1. Using appropriate quality control measures, 36,487 cells were chosen for downstream analysis. scRNA-seq studies identified that although VEGF signaling was not significantly altered in diabetic versus nondiabetic skin, phospholipase Cγ2 (PLCγ2) was downregulated. The significance of PLCγ2 in VEGF-mediated increase in endothelial cell metabolism and function was assessed in cultured human microvascular endothelial cells. In these cells, VEGF enhanced mitochondrial function, as indicated by elevation in oxygen consumption rate and extracellular acidification rate. The VEGF-dependent increase in cell metabolism was blunted in response to PLCγ2 inhibition. Follow-up rescue studies therefore focused on understanding the significance of VEGF therapy in presence or absence of endothelial PLCγ2 in type 1 (streptozotocin-injected) and type 2 (db/db) diabetic ischemic tissue. Nonviral topical tissue nanotransfection technology (TNT) delivery of CDH5 promoter-driven PLCγ2 open reading frame promoted the rescue of hindlimb ischemia in diabetic mice. Improvement of blood flow was also associated with higher abundance of VWF+/CD31+ and VWF+/SMA+ immunohistochemical staining. TNT-based gene delivery was not associated with tissue edema, a commonly noted complication associated with proangiogenic gene therapies. Taken together, our study demonstrates that TNT-mediated delivery of endothelial PLCγ2, as part of combination gene therapy, is effective in diabetic ischemic limb rescue.

Human adipose mesenchymal stem cells modulate inflammation and angiogenesis through exosomes

Stem cell-derived exosomes are efficient and safe therapeutic tools for transferring endogenous biological cargo or functional biomolecules for regenerative medicine. The regulation of inflammation and angiogenesis plays a pivotal role in wound healing and tissue regeneration. The purpose of this study was to investigate the anti-inflammatory and pro-angiogenic roles of human adipose mesenchymal stem cell-derived exosomes, focusing on the underlying mechanisms. Exosomes inhibited LPS-induced inflammation by activating ROCK1 and PTEN expression. Moreover, microRNAs (miR-132 and miR-146a) released from exosomes upregulated the expression of pro-angiogenic genes and promoted proliferation activity and tube formation in human umbilical vein endothelial cells. Exosomal effects were verified using ROCK1/PTEN inhibitors for anti-inflammation and miR-132/miR-146a inhibitors for pro-angiogenesis. Our findings suggest that exosomes exert anti-inflammatory effects by targeting the ROCK1/PTEN pathway and exhibit pro-angiogenic effects via delivery of miR-132 and miR-146a. Taken together, these results suggest that exosomes may be promising therapeutic candidates for curing diseases involved in inflammation and angiogenesis.

Abstract 163: Potential Therapeutic Effects Of Angiopoietin-2 Inhibition On Rescuing Hereditary Hemorrhagic Telangiectasia Associated Cerebrovascular Defects

Introduction: Hereditary Hemorrhagic Telangiectasia (HHT) is a vascular disease characterized by arteriovenous malformations (AVMs), which are abnormal, enlarged connections between arteries and veins that are prone to hemorrhage. AVMs often form in the brain and can lead to stroke. Mutations in endoglin (Eng), activin receptor-like kinase 1 (Alk1) or smad-related protein 4 (Smad4) cause HHT. Endothelial cell (EC) specific knock out of Alk1, Eng, or Smad4 in mice mimics HHT vascular defects, including AVM formation. We showed that Smad4 directly repressed transcription of the critical pro-angiogenic factor Angiopoietin-2 (Ang2) in ECs and that in Smad4-HHT mice, Ang2 /ANG2 levels are robustly and ectopically increased in ECs. By inhibiting Ang2, we prevented and rescued AVMs and normalized retinal vasculature. Since Smad4 is a central transcription factor in the TGF-β signaling pathway and a downstream effector of the Eng and Alk1 co-receptors, we hypothesized that Ang2 inhibition may universally normalize HHT vascular phenotypes. Methods: Blue latex dye perfusion methods were used to visualize and characterize cerebrovascular defects in HHT mouse models (n=5~7). RNA-seq was performed on isolated brain ECs (iBECs) to further define molecular phenotypic characteristics (n=5). In situ hybridization on brain slices (n=3) and ELISA on whole brain lysate (n=3~9) were performed to confirm elevated Ang2 RNA and protein levels. Ang2 inhibitor was delivered via intraperitoneal injection, followed by cerebrovascular assessments (n=5~9). Results: We found that brain AVMs are present in Smad4-, Alk1-, and Eng-HHT mutants, with all models displaying elevated Ang2 levels. RNA-seq of iBECs in Smad4-, Alk1-, and Eng-HHT models reveals 5502, 5307, and 1659 differentially expressed genes in respective mutants compared to controls. Data set comparisons revealed common, increased pro-angiogenic genes, which helped to define the HHT-specific angiogenic profile. Moreover, Ang2 inhibition decreased Smad4-HHT’s brain vessel diameter and Alk1-HHT’s brain vessel density, and prevented brain AVM formation in Eng-HHT mice. In conclusion, Ang2 inhibition partially normalized cerebrovascular defects in Smad4- and Alk1-HHT mice, and completely in Eng-HHT mice.

Distinctive Roles of YAP and TAZ in Human Endothelial Progenitor Cells Growth and Functions.

The hippo signaling pathway plays an essential role in controlling organ size and balancing tissue homeostasis. Its two main effectors, yes-associated protein (YAP) and WW domain-containing transcription regulator 1, WWTR1 or TAZ, have also been shown to regulate endothelial cell functions and angiogenesis. In this study, the functions of YAP and TAZ in human endothelial progenitor cells (EPCs) were investigated by a loss-of-function study using CRISPR/Cas9-mediated gene knockdown (KD). Depletion of either YAP or TAZ reduced EPC survival and impaired many of their critical functions, including migration, invasion, vessel-formation, and expression of pro-angiogenic genes. Notably, TAZ-KD EPCs exhibited more severe phenotypes in comparison to YAP-KD EPCs. Moreover, the conditioned medium derived from TAZ-KD EPCs reduced the survivability of human lung cancer cells and increased their sensitivity to chemotherapeutic agents. The overexpression of either wild-type or constitutively active TAZ rescued the impaired phenotypes of TAZ-KD EPCs and restored the expression of pro-angiogenic genes in those EPCs. In summary, we demonstrate the crucial role of Hippo signaling components, YAP and TAZ, in controlling several aspects of EPC functions that can potentially be used as a drug target to enhance EPC functions in patients.

Vascular Damage, Thromboinflammation, Plasmablast Activation, T-Cell Dysregulation and Pathological Histiocytic Response in Pulmonary Draining Lymph Nodes of COVID-19.

Although initial immunophenotypical studies on peripheral blood and bronchoalveolar lavage samples have provided a glimpse into the immunopathology of COVID-19, analyses of pulmonary draining lymph nodes are currently scarce. 22 lethal COVID-19 cases and 28 controls were enrolled in this study. Pulmonary draining lymph nodes (mediastinal, tracheal, peribronchial) were collected at autopsy. Control lymph nodes were selected from a range of histomorphological sequelae [unremarkable histology, infectious mononucleosis, follicular hyperplasia, non-SARS related HLH, extrafollicular plasmablast activation, non-SARS related diffuse alveolar damage (DAD), pneumonia]. Samples were mounted on a tissue microarray and underwent immunohistochemical staining for a selection of immunological markers and in-situ hybridization for Epstein Barr Virus (EBV) and SARS-CoV-2. Gene expression profiling was performed using the HTG EdgeSeq Immune Response Panel. Characteristic patterns of a dysregulated immune response were detected in COVID-19: 1. An accumulation of extrafollicular plasmablasts with a relative paucity or depletion of germinal centers. 2. Evidence of T-cell dysregulation demonstrated by immunohistochemical paucity of FOXP3+, Tbet+ and LEF1+ positive T-cells and a downregulation of key genes responsible for T-cell crosstalk, maturation and migration as well as a reactivation of herpes viruses in 6 COVID-19 lymph nodes (EBV, HSV). 3. Macrophage activation by a M2-polarized, CD163+ phenotype and increased incidence of hemophagocytic activity. 4. Microvascular dysfunction, evidenced by an upregulation of hemostatic (CD36, PROCR, VWF) and proangiogenic (FLT1, TEK) genes and an increase of fibrin microthrombi and CD105+ microvessels. Taken together, these findings imply widespread dysregulation of both innate and adoptive pathways with concordant microvascular dysfunction in severe COVID-19.

Multifaceted pathomolecular mechanism of a VWF large deletion involved in the pathogenesis of severe VWD

AbstractAn in-frame heterozygous large deletion of exons 4 through 34 of the von Willebrand factor (VWF) gene was identified in a type 3 von Willebrand disease (VWD) index patient (IP), as the only VWF variant. The IP exhibited severe bleeding episodes despite prophylaxis treatment, with a short VWF half-life after infusion of VWF/factor VIII concentrates. Transcript analysis confirmed transcription of normal VWF messenger RNA besides an aberrant deleted transcript. The IP endothelial colony-forming cells (ECFCs) exhibited a defect in the VWF multimers and Weibel-Palade bodies (WPBs) biogenesis, although demonstrating normal VWF secretion compared with healthy cells. Immunostaining of IP-ECFCs revealed subcellular mislocalization of WPBs pro-inflammatory cargos angiopoietin-2 (Ang2, nuclear accumulation) and P-selectin. Besides, the RNA-sequencing (RNA-seq) analysis showed upregulation of pro-inflammatory and proangiogenic genes, P-selectin, interleukin 8 (IL-8), IL-6, and GROα, copackaged with VWF into WPBs. Further, whole-transcriptome RNA-seq and subsequent gene ontology (GO) enrichment analysis indicated the most enriched GO-biological process terms among the differentially expressed genes in IP-ECFCs were regulation of cell differentiation, cell adhesion, leukocyte adhesion to vascular endothelial, blood vessel morphogenesis, and angiogenesis, which resemble downstream signaling pathways associated with inflammatory stimuli and Ang2 priming. Accordingly, our functional experiments exhibited an increased endothelial cell adhesiveness and interruption in endothelial cell–cell junctions of the IP-ECFCs. In conclusion, the deleted VWF has a dominant-negative impact on multimer assembly and the biogenesis of WPBs, leading to altered trafficking of their pro-inflammatory cargos uniquely, which, in turn, causes changes in cellular signaling pathways, phenotype, and function of the endothelial cells.

Retinal Diseases Regulated by Hypoxia-Basic and Clinical Perspectives: A Comprehensive Review.

In recent years, the number of patients with age-related macular degeneration (AMD) is increasing worldwide along with increased life expectancy. Currently, the standard treatment for wet-AMD is intravitreal injection of anti-vascular endothelial growth factor (VEGF) drugs. The upstream of VEGF is hypoxia-inducible factor (HIF), a master regulator of hypoxia-responsive genes responsive to acute and chronic hypoxia. HIF activation induces various pathological pro-angiogenic gene expressions including VEGF under retinal hypoxia, ultimately leading to the development of ocular ischemic neovascular diseases. In this regard, HIF is considered as a promising therapeutic target in ocular ischemic diseases. In clinical ophthalmology, abnormal hypofluorescent areas have been detected in the late-phase of indocyanine green angiography, which are thought to be lipid deposits at the level of Bruch’s membrane to choriocapillaris in vitreoretinal diseases. These deposits may interfere with the oxygen and nutrients that should be supplied to the retinal pigment epithelium, and that HIF/VEGF is highly suspected to be expressed in the hypoxic retinal pigment epithelium, leading to neovascularization. In this review, we comprehensively summarize pathophysiology of AMD-related ocular diseases with the HIF/VEGF pathway from basic and clinic researches with recent findings.

SIRT1-mediated deacetylation of FOXO3a transcription factor supports pro-angiogenic activity of interferon-deficient tumor-associated neutrophils.

Angiogenesis plays an important role during tumor growth and metastasis. We could previously show that Type I interferon (IFN)-deficient tumor-associated neutrophils (TANs) show strong pro-angiogenic activity, and stimulate tumor angiogenesis and growth. However, the exact mechanism responsible for their pro-angiogenic shift is not clear. Here, we set out to delineate the molecular mechanism and factors regulating pro-angiogenic properties of neutrophils in the context of Type I IFN availability. We demonstrate that neutrophils from IFN-deficient (Ifnar1-/- ) mice efficiently release pro-angiogenic factors, such as VEGF, MMP9 or BV8, and thus significantly support the vascular normalization of tumors by increasing the maturation of perivascular cells. Mechanistically, we could show here that the expression of pro-angiogenic factors in neutrophils is controlled by the transcription factor forkhead box protein O3a (FOXO3a), which activity depends on its post-translational modifications, such as deacetylation or phosphorylation. In TANs isolated from Ifnar1-/- mice, we observe significantly elevated SIRT1, resulting in SIRT1-mediated deacetylation of FOXO3a, its nuclear retention and activation. Activated FOXO3a supports in turn the transcription of pro-angiogenic genes in TANs. In the absence of SIRT1, or after its inhibition in neutrophils, elevated kinase MEK/ERK and PI3K/AKT activity is observed, leading to FOXO3a phosphorylation, cytoplasmic transfer and inactivation. In summary, we have found that FOXO3a is a key transcription factor controlling the angiogenic switch of neutrophils. Post-translational FOXO3a modifications regulate its transcriptional activity and, as a result, the expression of pro-angiogenic factors supporting development of vascular network in growing tumors. Therefore, targeting FOXO3a activity could provide a novel strategy of antiangiogenic targeted therapy for cancer.

Isopimpinellin extends antiangiogenic effect through overexpression of miR-15b-5p and downregulating angiogenic stimulators.

Angiogenesis is the formation of new blood vessels from an existing vasculature through a series of processes such as activation, proliferation, and directed migration of endothelial cells. Angiogenesis is instrumental in the metastatic spread of tumors. Isopimpinellin, a furanocoumarin group of phytochemicals, is an anticarcinogenic agent. However, no studies have proven its antiangiogenic effects. The current study thus aimed to screen the antiangiogenic effect of isopimpinellin. Human Umblical Vein Endothelial Cell (HUVEC) as an in vitro model and zebrafish embryos as an in vivo model was used in this study. The experimental results showed that isopimpinellin effectively inhibited HUVEC proliferation, invasion, migration, and tube formation, which are the key steps in angiogenesis by markedly suppressing the expression of pro-angiogenic genes VEGF, AKT, and HIF-1α. In addition, isopimpinellin exerts its anti-angiogenic effect through the regulation of miR-15b-5p and miR-542-3p. Furthermore, in zebrafish embryos, isopimpinellin inhibited the development of intersegmental vessels (ISVs) through the significant downregulation of all pro-angiogenic genes vegf, vegfr2, survivin, angpt-1, angpt-2, and tie-2. Collectively, these experimental findings offer novel insights into the antiangiogenic nature of isopimpinellin and open new avenues for therapeutic approaches.

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.

Bufalin suppresses tumour microenvironment-mediated angiogenesis by inhibiting the STAT3 signalling pathway

BackgroundAntiangiogenic therapy has increasingly become an important strategy for the treatment of colorectal cancer. Recent studies have shown that the tumour microenvironment (TME) promotes tumour angiogenesis. Bufalin is an active antitumour compound whose efficacy has been indicated by previous studies. However, there are very few studies on the antiangiogenic effects of bufalin.MethodsHerein, human umbilical vein endothelial cell (HUVEC) tube formation, migration and adhesion tests were used to assess angiogenesis in vitro. Western blotting and quantitative PCR were used to detect relevant protein levels and mRNA expression levels. A subcutaneous xenograft tumour model and a hepatic metastasis model were established in mice to investigate the influence of bufalin on angiogenesis mediated by the TME in vivo.ResultsWe found that angiogenesis mediated by cells in the TME was significantly inhibited in the presence of bufalin. The results demonstrated that the proangiogenic genes in HUVECs, such as VEGF, PDGFA, E-selectin and P-selectin, were downregulated by bufalin and that this downregulation was mediated by inhibition of the STAT3 pathway. Overexpression of STAT3 reversed the inhibitory effects of bufalin on angiogenesis. Furthermore, there was little reduction in angiogenesis when bufalin directly acted on the cells in the tumour microenvironment.ConclusionOur findings demonstrate that bufalin suppresses tumour microenvironment-mediated angiogenesis by inhibiting the STAT3 signalling pathway in vascular endothelial cells, revealing that bufalin may be used as a new antiangiogenic adjuvant therapy medicine to treat colorectal cancer.

MP39-03 INTEGRATION OF NEUROPILIN 1, REGULATOR OF G PROTEIN SIGNALLING 5, AND FORKHEAD BOX M1 EXPRESSION AND TUMOUR NECROSIS AS A PROGNOSTIC CLASSIFIER BASED ON MOLECULAR SUBTYPES OF CLEAR CELL RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology I (MP39)1 Sep 2021MP39-03 INTEGRATION OF NEUROPILIN 1, REGULATOR OF G PROTEIN SIGNALLING 5, AND FORKHEAD BOX M1 EXPRESSION AND TUMOUR NECROSIS AS A PROGNOSTIC CLASSIFIER BASED ON MOLECULAR SUBTYPES OF CLEAR CELL RENAL CELL CARCINOMA Takashi Yoshida, Chisato Ohe, Junichi Ikeda, Ryoichi Saito, Haruyuki Ohsugi, Motohiko Sugi, Hidefumi Kinoshita, and Tadashi Matsuda Takashi YoshidaTakashi Yoshida More articles by this author , Chisato OheChisato Ohe More articles by this author , Junichi IkedaJunichi Ikeda More articles by this author , Ryoichi SaitoRyoichi Saito More articles by this author , Haruyuki OhsugiHaruyuki Ohsugi More articles by this author , Motohiko SugiMotohiko Sugi More articles by this author , Hidefumi KinoshitaHidefumi Kinoshita More articles by this author , and Tadashi MatsudaTadashi Matsuda More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002054.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Molecular mechanisms of progression of clear cell renal cell carcinoma (ccRCC) have been proven with recent genomic or transcriptional analyses. However, it is still difficult to apply these analyses to daily clinical practice owing to economical and practical issues. Here, we established a pathology-based prognostic classification based on the well-validated transcriptional classifier, ClearCorde34, in ccRCC. METHODS: Three-hundred forty-two cases with available tissue were identified and randomly divided into the discovery cohort (n=138) and the validation cohort (n=204). Levels of mRNA were quantified using the Digital Analyzer, and the ccA/ccB subtypes were determined. Histological and immunohistochemistry (IHC) analyses were subsequently performed to establish the pathology-based classification based on the mRNA levels. Finally, the prognostic ability of the new classifier was evaluated in both the discovery and validation cohorts. RESULTS: Of 138 cases in the discovery cohort, 78 (56.5%) and 60 (43.5%) were assigned to the ccA and ccB subtypes, respectively. Proangiogenic genes, neuropilin 1 (NRP1) and regulator of G protein signalling 5 (RGS5), were especially overexpressed in all ccRCC samples and were enriched in ccA-assigned tumours. Histologically, tumour necrosis and the sarcomatoid feature were associated with the ccB subtype. In IHC analyses, expression of NRP1, RGS5, and Forkhead box M1 (FOXM1), an epithelial-mesenchymal transition-related factor, significantly correlated with the ccA/ccB subtypes. Combining these three IHC factors and tumour necrosis, we developed the IHC/histology-based classifier, which showed good concordance with the ClearCode34 classifier with an accuracy of 0.80. The established classification significantly stratified relapse-free, cancer-specific, and overall survival rates in both the discovery and validation cohorts. CONCLUSIONS: The novel molecular pathology classifier integrating NRP1, RGS5, FOXM1, and tumour necrosis may enable the stratification of oncological outcomes for patients with ccRCC. Source of Funding: None © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e704-e705 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takashi Yoshida More articles by this author Chisato Ohe More articles by this author Junichi Ikeda More articles by this author Ryoichi Saito More articles by this author Haruyuki Ohsugi More articles by this author Motohiko Sugi More articles by this author Hidefumi Kinoshita More articles by this author Tadashi Matsuda More articles by this author Expand All Advertisement Loading ...

Angiogenic gene networks are dysregulated in opioid use disorder: evidence from multi-omics and imaging of postmortem human brain.

Opioid use disorder (OUD) is a public health crisis in the U.S. that causes over 50 thousand deaths annually due to overdose. Using next-generation RNA sequencing and proteomics techniques, we identified 394 differentially expressed (DE) coding and long noncoding (lnc) RNAs as well as 213 DE proteins in Brodmann Area 9 of OUD subjects. The RNA and protein changes converged on pro-angiogenic gene networks and cytokine signaling pathways. Four genes (LGALS3, SLC2A1, PCLD1, VAMP1) were dysregulated in both RNA and protein. Dissecting these DE genes and networks, we found cell type specific effects with enrichment in astrocyte, endothelial, and microglia correlated genes. Weighted-genome correlation network analysis (WGCNA) revealed cell type correlated networks including an astrocytic/endothelial/microglia network involved in angiogenic cytokine signaling as well as a neuronal network involved in synaptic vesicle formation. In addition, using ex vivo magnetic resonance imaging, we identified increased vascularization in postmortem brains from a subset of subjects with OUD. This is the first study integrating dysregulation of angiogenic gene networks in OUD with qualitative imaging evidence of hypervascularization in postmortem brain. Understanding the neurovascular effects of OUD is critical in this time of widespread opioid use.

UTP is a regulator of in vitro and in vivo angiogenic properties of cardiac adipose-derived stem cells.

The ability of cardiac adipose-derived stem cells (cADSC) to differentiate into multiple cell types has opened new perspectives in cardiac cell-based regenerative therapies. P2Y nucleotide receptors have already been described as regulators of adipogenic differentiation of cADSC and bone marrow-derived stem cells. In this study, we defined UTP as a regulator of cADSC endothelial differentiation. A daily UTP stimulation of cADSC during endothelial predifferentiation increased their capacity to form an endothelial network in matrigel. Additionally, pro-angiogenic UTP target genes such as epiregulin and hyaluronan synthase-1 were identified in predifferentiated cADSC by RNA sequencing experiments. Their regulation by UTP was confirmed by qPCR and ELISA experiments. We then evaluated the capacity of UTP-treated predifferentiated cADSC to increase post-ischemic revascularization in mice subjected to left anterior descending artery ligation. Predifferentiated cADSC treated or not with UTP were injected in the periphery of the infarcted zone, 3days after ligation. We observed a significant increase of capillary density 14 and 30days after UTP-treated predifferentiated cADSC injection, correlated with a reduction of cardiac fibrosis. This revascularization increase was not observed after injection of UTP-treated cADSC deficient for UTP and ATP nucleotide receptor P2Y2. The present study highlights the P2Y2 receptor as a regulator of cADSC endothelial differentiation and as a potential target for the therapeutic use of cADSC in post-ischemic heart revascularization.

Endothelial cell PHD2-HIF1α-PFKFB3 contributes to right ventricle vascular adaptation in pulmonary hypertension.

Humans and animals with pulmonary hypertension (PH) show right ventricular (RV) capillary growth, which positively correlates with overall RV hypertrophy. However, molecular drivers of RV vascular augmentation in PH are unknown. Prolyl hydroxylase (PHD2) is a regulator of hypoxia-inducible factors (HIFs), which transcriptionally activates several proangiogenic genes, including the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). We hypothesized that a signaling axis of PHD2-HIF1α-PFKFB3 contributes to adaptive coupling between the RV vasculature and tissue volume to maintain appropriate vascular density in PH. We used design-based stereology to analyze endothelial cell (EC) proliferation and the absolute length of the vascular network in the RV free wall, relative to the tissue volume in mice challenged with hypoxic PH. We observed increased RV EC proliferation starting after 6 h of hypoxia challenge. Using parabiotic mice, we found no evidence for a contribution of circulating EC precursors to the RV vascular network. Mice with transgenic deletion or pharmacological inhibition of PHD2, HIF1α, or PFKFB3 all had evidence of impaired RV vascular adaptation following hypoxia PH challenge. PHD2-HIF1α-PFKFB3 contributes to structural coupling between the RV vascular length and tissue volume in hypoxic mice, consistent with homeostatic mechanisms that maintain appropriate vascular density. Activating this pathway could help augment the RV vasculature and preserve RV substrate delivery in PH, as an approach to promote RV function.

O6 Analysis of angiogenic gene profiling after E-selectin + stem cell therapy in murine ischemic limb

Abstract Introduction There remains a paucity of novel therapeutics for limb salvage in patients with critical limb ischemia (CLI) for whom revascularization procedures have failed and amputation is imminent. We have shown that E-selectin+/Mesenchymal Stem Cell (MSC) injections into the ischemic limb tissue of a CLI mouse model improves revascularization and limb function. Thus, we sought to determine a mechanism of action for E-selectin+/MSC’s pro-angiogenic and tissue salvage properties. Methods MSC were extracted from donor mice bone marrow and subsequently engineered via viral transduction with E-selectin-ires-GFP/AAV and GFP/AAV (control) to create E-selectin-GFP+/MSC vs GFP+/MSC. Intramuscular injections of E-selectin-GFP+/MSC, GFP+/MSC, or PBS were performed in a mouse model of hindlimb ischemia. Laser doppler imaging (LDI), confocal laser microscopy, and treadmill exhaustion test were utilized to determine neovascularization and limb function. RNA extraction from engineered MSC (E-selectin-GFP+/MSC vs GFP+/MSC) and ischemic hindlimb tissues treated with E-selectin-GFP+/MSC vs GFP+/MSC was performed, followed by RT2 Profiler PCR Array analysis of 84 genes involved in angiogenesis. GFP+/MSC treated hindlimb tissue served as control. Student’s t-test or ANOVA was utilized to compare means and significance set at P < 0.05. Results Compared with GFP+/MSC and PBS, treatment with E-selectin-GFP+/MSC increased ischemic leg LDI reperfusion (54% vs. 39% vs. 22%, P < 0.001), treadmill distance traversed (162 m vs. 111 m vs. 110 m, P < 0.01) and ischemic mouse footpad vessel density (23% vs. 14% vs. 14%, P < 0.01). RT2 Profiler PCR Array demonstrated pro-angiogenic gene upregulation occurred in 7 genes (Csf3, Cxcl2, Cxcl5, Serpine1, F2, Lep, Tbx1, Table I.) in E-selectin-GFP+/MSC treated ischemic leg tissue while tumour necrosis factor (TNF) was found to be downregulated, when compared with GFP+/MSC treated tissues. Of these 7 upregulated genes, CXCl2, F2, Leptin and T-box1 (Table I.) are likely produced by E-selectin-GFP+/MSC, as analysis of cellular gene expression profiles of E-selectin-GFP+/MSC also revealed upregulation by 2-fold or more in these factors when compared to GFP+/MSC. Validation of gene functions in-vivo are under investigation. Conclusion Stem cell therapy using E-selectin-GFP+/MSC, in a murine model of CLI, confers both augmented postnatal neovascularization and increased limb function. The pro-angiogenic and pro-repair effects are likely mediated by upregulation of a panel of chemokines/cytokines and down-regulation of TNF in ischemic tissues treated with E-selectin-GFP+/MSC.

Dual l-Carnosine/Aloe vera Nanophytosomes with Synergistically Enhanced Protective Effects against Methylglyoxal-Induced Angiogenesis Impairment.

Microvascular complications are among the major outcomes of patients with type II diabetes mellitus, which are the consequences of impaired physiological functioning of small blood vessels and angiogenic responses in these patients. Overproduction and accumulation of methylglyoxal (MGO), a highly reactive dicarbonyl byproduct of glycolysis pathway, has been acclaimed as the main inducer of impaired angiogenic responses and microvascular dysfunction in diabetic patients with uncontrolled hyperglycemia. Hence, an effective approach to overcome diabetes-associated microvascular complications is to neutralize the deleterious activity of enhanced the concentration of MGO in the body. Owing to the glycation inhibitory activity of Aloe vera whole extract, and capability of l-carnosine, an endogenous dipeptide, in attenuating MGO's destructive activity, we examined whether application of a combination of l-carnosine and A. vera could be an effective way of synergistically weakening this reactive dicarbonyl's impaired angiogenic effects. Additionally, overcoming the poor cellular uptake and internalization of l-carnosine and A. vera, a nanophytosomal formulation of the physical mixture of two compounds was also established. Although l-carnosine and A. vera at whole studied combination ratios could synergistically enhance viability of human umbilical vein endothelial cells (HUVECs) treated with MGO, the 25:1 w/w ratio was the most effective one among the others (27 ± 0.5% compared to 12 ± 0.3 to 18 ± 0.4%; F (4, 15) = 183.9, P < 0.0001). Developing dual nanophytosomes of l-carnosine/A. vera (25:1) combination ratio, we demonstrated superiority of the nanophytosomal formulation in protecting HUVECs against MGO-induced toxicity following a 24-72 h incubation period (17.3, 15.8, and 12.4% respectively). Moreover, 500 μg/mL concentration of dual l-carnosine/A. vera nanophytosomes exhibited a superior free radical scavenging potency (63 ± 4 RFU vs 83 ± 5 RFU; F (5, 12) = 54.81, P < 0.0001) and nitric oxide synthesizing capacity (26.11 ± 0.19 vs 5.1 ± 0.33; F (5, 12) = 2537, P < 0.0001) compared to their physical combination counterpart. Similarly, 500 μg/mL dual l-carnosine/A. vera nanophytosome-treated HUVECs demonstrated a superior tube formation capacity (15 ± 3 vs 2 ± 0.3; F (5, 12) = 30.87, P < 0.001), wound scratch healing capability (4.92 ± 0.3 vs 3.07 ± 0.3 mm/h; F (5, 12) = 39.21, P < 0.0001), and transwell migration (586 ± 32 vs 394 ± 18; F (5, 12) = 231.8, P < 0.001) and invasion (172 ± 9 vs 115 ± 5; F (5, 12) = 581.1, P < 0.0001) activities compared to the physical combination treated ones. Further confirming the proangiogenic activity of the dual l-carnosine/A. vera nanophytosomes, a significant shift toward expression of proangiogenic genes including HIF-1α, VEGFA, bFGF, KDR, and Ang II was reported in treated HUVECs. Overall, dual l-carnosine/A. vera nanophytosomes could be a potential candidate for attenuating type II DM-associated microvascular complications with an impaired angiogenesis background.

RNA-binding protein SAMD4A inhibits breast tumor angiogenesis by modulating the balance of angiogenesis program.

Tumor‐induced angiogenesis is important for further progression of solid tumors. The initiation of tumor angiogenesis is dictated by a shift in the balance between proangiogenic and antiangiogenic gene expression programs. However, the potential mechanism controlling the expression of angiogenesis‐related genes in the tumor cells, especially the process mediated by RNA‐binding protein (RBP) remains unclear. SAMD4A is a conserved RBP across fly to mammals, and is believed to play an important role in controlling gene translation and stability. In this study, we identified the potential role of SAMD4A in modulating angiogenesis‐related gene expression and tumor progression in breast cancer. SAMD4A expression was repressed in breast cancer tissues and cells and low SAMD4A expression in human breast tumor samples was strongly associated with poor survival of patients. Overexpression of SAMD4A inhibited breast tumor angiogenesis and caner progression, whereas knockdown of SAMD4A demonstrated a reversed effect. Mechanistically, SAMD4A was found to specifically destabilize the proangiogenic gene transcripts, including C‐X‐C motif chemokine ligand 5 (CXCL5), endoglin (ENG), interleukin 1β (IL1β), and angiopoietin 1 (ANGPT1), by directly interacting with the stem‐loop structure in the 3′ untranslated region (3′UTR) of these mRNAs through its sterile alpha motif (SAM) domain, resulting in the imbalance of angiogenic genes expression. Collectively, our results suggest that SAMD4A is a novel breast tumor suppressor that inhibits tumor angiogenesis by specifically downregulating the expression of proangiogenic genes, which might be a potential antiangiogenic target for breast cancer therapy.

Integration of NRP1, RGS5, and FOXM1 expression, and tumour necrosis, as a postoperative prognostic classifier based on molecular subtypes of clear cell renal cell carcinoma.

Molecular mechanisms of progression of clear cell renal cell carcinoma (ccRCC) have been proven with recent genomic or transcriptional analyses. However, it is still difficult to apply these analyses to daily clinical practice owing to economical and practical issues. Here, we established a pathology‐based, postoperative prognostic classification based on the well‐validated transcriptional classifier, ClearCode34, in ccRCC. A total of 342 cases with available tissue were identified and randomly allocated into a discovery cohort (n = 138) and a validation cohort (n = 204). Levels of mRNA were quantified using a nCounter Digital Analyzer, and the ccA/ccB subtypes were determined. Histological and immunohistochemistry (IHC) analyses were subsequently performed to establish a pathology‐based classification based on the mRNA levels. Finally, the prognostic ability of the new classifier was evaluated in both the discovery and validation cohorts. Of 138 cases in the discovery cohort, 78 (56.5%) and 60 (43.5%) were assigned to the ccA and ccB subtypes, respectively. Proangiogenic genes, neuropilin 1 (NRP1) and regulator of G protein signalling 5 (RGS5), were especially overexpressed in all ccRCC samples and were enriched in ccA‐assigned tumours. Histologically, tumour necrosis and the sarcomatoid feature were associated with the ccB subtype. In IHC analyses, expression of NRP1, RGS5, and forkhead box M1 (FOXM1), an epithelial–mesenchymal transition‐related factor, significantly correlated with the ccA/ccB subtypes. Combining these three IHC factors and tumour necrosis, we developed the IHC/histology‐based classifier, which showed good concordance with the ClearCode34 classifier with an accuracy of 0.80. The established classification significantly stratified relapse‐free, cancer‐specific, and overall survival rates in both the discovery and validation cohorts. The novel molecular pathology classifier integrating NRP1, RGS5, FOXM1, and tumour necrosis may enable the stratification of oncological outcomes for patients with ccRCC undergoing resection surgery.