Antiangiogenic Gene Research Articles

EP.TH.729Spontaneous Complete Regression of Sacroma: A Case Report And Review of Literature

Abstract Spontaneous complete regression (SR) of cancer is complete or partial disappearance of the cancer without any treatment which is very rare(1). Case Report 58 year old female, with a palpable right breast lump from the chest wall at 7o clock position. It was 25mm in size, axillae demonstrated no LAD. Imaging was performed which showed a suspicious lesion with malignant features,core biopsy done. It showed pleomorphic spindled and epitheloid cells. Immunohistochemistry was negative for AE1/AE3,MNF116,CK19,CK7,P63,CK14,CK5/6 was diagnosed as sarcoma with a possibility of phyllodes tumour. Plan was made in MDT to discuss in regional sarcoma meeting and staging CT which was normal. Sarcoma MDT recommended surgery, planned for wide local excision. On the day of surgery patient came for wire localisation, however no tumor noted on imaging and patient also confirmed the same. Patient had CT and MRI which revealed no abnormalities. This was discussed in local and central MDT, advised imaging after 3 and 6 months. Discussion Spontaneous regression of breast sarcoma in Pub med yielded no cases. This process has not been clearly explained yet. Several mechanisms have been suggested for spontaneous regression, including immune modulation, withdrawal of carcinogens, infection, apoptosis, antiangiogenic and maturation mechanisms gene therapy, vaccine-mediated mechanisms among many. Conclusion This is the first case of SA of primary breast sarcoma to our knowledge and our main aim to present this case is due to its rarity.

Angiogenic Potential of VEGF Mimetic Peptides for the Biofunctionalization of Collagen/Hydroxyapatite Composites.

Currently, the focus on bioinspired concepts for the development of tissue engineering constructs is increasing. For this purpose, the combination of collagen (Coll) and hydroxyapatite (HA) comes closest to the natural composition of the bone. In order to confer angiogenic properties to the scaffold material, vascular endothelial growth factor (VEGF) is frequently used. In the present study, we used a VEGF mimetic peptide (QK) and a modified QK-peptide with a poly-glutamic acid tag (E7-QK) to enhance binding to HA, and analyzed in detail binding efficiency and angiogenic properties. We detected a significantly higher binding efficiency of E7-QK peptides to hydroxyapatite particles compared to the unmodified QK-peptide. Tube formation assays revealed similar angiogenic functions of E7-QK peptide (1µM) as induced by the entire VEGF protein. Analyses of gene expression of angiogenic factors and their receptors (FLT-1, KDR, HGF, MET, IL-8, HIF-1α, MMP-1, IGFBP-1, IGFBP-2, VCAM-1, and ANGPT-1) showed higher expression levels in HUVECs cultured in the presence of 1 µM E7-QK and VEGF compared to those detected in the negative control group without any angiogenic stimuli. In contrast, the expression of the anti-angiogenic gene TIMP-1 showed lower mRNA levels in HUVECs cultured with E7-QK and VEGF. Sprouting assays with HUVEC spheroids within Coll/HA/E7-QK scaffolds showed significantly longer sprouts compared to those induced within Coll/HA/QK or Coll/HA scaffolds. Our results demonstrate a significantly better functionality of the E7-QK peptide, electrostatically bound to hydroxyapatite particles compared to that of unmodified QK peptide. We conclude that the used E7-QK peptide represents an excellently suited biomolecule for the generation of collagen/hydroxyapatite composites with angiogenic properties.

Coronary artery disease ameliorates extracellular vesicle lncRNA PUNISHER regulates angiogenic response andendothelial cells function via NFkB-dependent mechanism

Abstract Augmenting evidence indicates that long noncoding RNAs (lncRNAs) are playing a crucial role in diverse cellular/pathological processes. Intercellular transfer of extracellular vesicles (EVs) transmitted lncRNA regulates vascular health and diseases. However, whether lncRNA expression in EVs is regulated in patients with coronary artery disease, is unknown. A PCR-based lncRNA array analysis revealed that EV-PUNISHER was significantly upregulated in patients with CAD (n=221) compared to healthy subjects. Our data showed that PUNISHER is the most upregulated lncRNA in CAD in accordance with other highly expressed lncRNAs. To examine the specific role of PUNISHER in EC phenotypic regulation, siRNA-mediated silencing in ECs revealed that depletion of PUNISHER suppresses the migration and proliferation of ECs. Moreover, depletion of PUNISHER decreases cell survival by reducing cell viability and proliferation through increased apoptosis and cytotoxicity. In order to investigate EC function in PUNISHER depleted cells, in vitro sprouting and tube formation assay revealed that PUNISHER is an important mediator EC functions, which acts as an anti-angiogenic factor. In vitro atherosclerotic stimuli increased PUNISHER expression in EV/EC in a dose-dependent way. Microarray analysis identified a series of pro- and anti-angiogenic genes as well genes directly involved in cell viability that are differentially regulated. We confirmed that PUNISHER is incorporated into endothelial microvesicles (EMVs) and transferred to recipient cells by using different experiments. By using lncRNA-FISH and vesicle degradation assays, we showed that PUNISHER is incorporated into EMVs, augmented recipient EC function in vitro upon transfer via EMVs. To examine whether EMV-PUNISHER promotes the EC function, different in vitro functional experiments with ECs confirmed that PUNISHER is an important regulator. Mass spectrometry analysis has revealed EMV contains numerous proteins, including RNA binding proteins such as hnRNPU. By using RNA-pulldown and RNA-IP, we confirmed that PUNISHER interacts with hnRNPU, which facilitates packaging into EMV prior to transfer to recipient EC. The interaction acts as an important positive regulator of cell viability and survival of recipient cells, identified using functional assay. Transcription factor array has shown that PUNISHER regulates NFkB to control cellular viability and apoptosis. A murine re-endothelialization model after injection of 1×107 EVs or bulk ncRNAs revealed that EV-PUNISHER promotes reendothelialization. Ex vivo internalization/uptake of PKH-67-labeled EVs revealed that EVs are detectable in the cross-section of EV-perfused carotid artery. Our study unveiled for the first time that EV-PUNISHER exerts its function in endothelial cells which might be beneficial in cardiovascular pathologies where the endothelial function is dysregulated. Our data indicate that PUNISHER can be targeted to develop targeted therapeutics. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): German Research Foundation (DFG); German Society of Cardiology (DGK)

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.

Fibroblast GATA-4 and GATA-6 promote myocardial adaptation to pressure overload by enhancing cardiac angiogenesis

Heart failure due to high blood pressure or ischemic injury remains a major problem for millions of patients worldwide. Despite enormous advances in deciphering the molecular mechanisms underlying heart failure progression, the cell-type specific adaptations and especially intercellular signaling remain poorly understood. Cardiac fibroblasts express high levels of cardiogenic transcription factors such as GATA-4 and GATA-6, but their role in fibroblasts during stress is not known. Here, we show that fibroblast GATA-4 and GATA-6 promote adaptive remodeling in pressure overload induced cardiac hypertrophy. Using a mouse model with specific single or double deletion of Gata4 and Gata6 in stress activated fibroblasts, we found a reduced myocardial capillarization in mice with Gata4/6 double deletion following pressure overload, while single deletion of Gata4 or Gata6 had no effect. Importantly, we confirmed the reduced angiogenic response using an in vitro co-culture system with Gata4/6 deleted cardiac fibroblasts and endothelial cells. A comprehensive RNA-sequencing analysis revealed an upregulation of anti-angiogenic genes upon Gata4/6 deletion in fibroblasts, and siRNA mediated downregulation of these genes restored endothelial cell growth. In conclusion, we identified a novel role for the cardiogenic transcription factors GATA-4 and GATA-6 in heart fibroblasts, where both proteins act in concert to promote myocardial capillarization and heart function by directing intercellular crosstalk.

High Glucose Treatment Limits Drosha Protein Expression and Alters AngiomiR Maturation in Microvascular Primary Endothelial Cells via an Mdm2-dependent Mechanism.

Diabetes promotes an angiostatic phenotype in the microvascular endothelium of skeletal muscle and skin. Angiogenesis-related microRNAs (angiomiRs) regulate angiogenesis through the translational repression of pro- and anti-angiogenic genes. The maturation of micro-RNA (miRs), including angiomiRs, requires the action of DROSHA and DICER proteins. While hyperglycemia modifies the expression of angiomiRs, it is unknown whether high glucose conditions alter the maturation process of angiomiRs in dermal and skeletal muscle microvascular endothelial cells (MECs). Compared to 5 mM of glucose, high glucose condition (30 mM, 6–24 h) decreased DROSHA protein expression, without changing DROSHA mRNA, DICER mRNA, or DICER protein in primary dermal MECs. Despite DROSHA decreasing, high glucose enhanced the maturation and expression of one angiomiR, miR-15a, and downregulated an miR-15a target: Vascular Endothelial Growth Factor-A (VEGF-A). The high glucose condition increased Murine Double Minute-2 (MDM2) expression and MDM2-binding to DROSHA. Inhibition of MDM2 prevented the effects evoked by high glucose on DROSHA protein and miR-15a maturation in dermal MECs. In db/db mice, blood glucose was negatively correlated with the expression of skeletal muscle DROSHA protein, and high glucose decreased DROSHA protein in skeletal muscle MECs. Altogether, our results suggest that high glucose reduces DROSHA protein and enhances the maturation of the angiostatic miR-15a through a mechanism that requires MDM2 activity.

TARBP2 promotes tumor angiogenesis and metastasis by destabilizing antiangiogenic factor mRNAs.

Tumor angiogenesis is a crucial step in the further growth and metastasis of solid tumors. However, its regulatory mechanism remains unclear. Here, we showed that TARBP2, an RNA‐binding protein, played a role in promoting tumor‐induced angiogenesis both in vitro and in vivo through degrading the mRNAs of antiangiogenic factors, including thrombospondin1/2 (THBS1/2), tissue inhibitor of metalloproteinases 1 (TIMP1), and serpin family F member 1 (SERPINF1), by targeting their 3′untranslated regions (3′UTRs). Overexpression of TARBP2 promotes tumor cell–induced angiogenesis, while its knockdown inhibits tumor angiogenesis. Clinical cohort analysis revealed that high expression level of TARBP2 was associated with poor survival of lung cancer and breast cancer patients. Mechanistically, TARBP2 physically interacts with the stem‐loop structure located in the 3′UTR of antiangiogenic transcripts, leading to mRNA destabilization by the dsRNA‐binding domains 1/2 (dsRBDs1/2). Notably, the expression level of TARBP2 in human tumor tissue is negatively correlated with the expression of antiangiogenic factors, including THBS1/2, and brain‐specific angiogenesis inhibitor 1 (BAI1). Moreover, TARBP2 expression is strongly associated with tumor angiogenesis in a group of human lung cancer samples. Collectively, our results highlight that TARBP2 is a novel tumor angiogenesis regulator that could promote tumor angiogenesis by selectively downregulating antiangiogenic gene expression.

Viral-Vector-Delivered Anti-Angiogenic Therapies to the Eye

Pathological vessel growth harms vision and may finally lead to vision loss. Anti-angiogenic gene therapy with viral vectors for ocular neovascularization has shown great promise in preclinical studies. Most of the studies have been conducted with different adeno-associated serotype vectors. In addition, adeno- and lentivirus vectors have been used. Therapy has been targeted towards blocking vascular endothelial growth factors or other pro-angiogenic factors. Clinical trials of intraocular gene therapy for neovascularization have shown the treatment to be safe without severe adverse events or systemic effects. Nevertheless, clinical studies have not proceeded further than Phase 2 trials.

Oral Contraceptive Steroids Promote Papillary Thyroid Cancer Metastasis by Targeting Angiogenesis and Epithelial-Mesenchymal Transition.

Thyroid cancer is the most prevalent type of endocrine malignancy with the highest incidence rate among women under 45 years old. Ethinylestradiol (EE) and levonorgestrel (LNG) are two steroid components of low-dose oral contraceptives used all over the world. In this study, we aimed to examine the possible effects of the combination of these two steroids on metastasis and angiogenic factors in BCPAP papillary thyroid cancer (PTC) cell line. After treatment of BCPAP cells with the combination of 20 nM EE and 90 nM LNG, mRNA expression levels of long noncoding RNAs HOTAIR and MALAT1, angiogenic and antiangiogenic gene markers VEGFA and THBS1, and epithelial-mesenchymal transition (EMT) biomarkers CDH1, CDH2, FN1, and VIM were analyzed by real-time PCR. Additionally, the protein expression of VEGFA was semiquantified by Western blotting. Results showed that the combination of LNG and EE significantly elevated the level of VEGFA protein and mRNA expression of VEGFA, MALAT1, HOTAIR, CDH2, FN1, and VIM genes while decreased CDH1 gene expression but had no marked effect on the expression of THBS1 gene in comparison with the control group. Also, our results suggest that LNG and EE may increase the metastatic and migratory properties of BCPAP cells via modulating angiogenic and EMT biomarkers. These data may highlight the potential of exogenous steroids in the advancement of PTC tumors.

Roquin2 suppresses breast cancer progression by inhibiting tumor angiogenesis via selectively destabilizing proangiogenic factors mRNA.

Tumor angiogenesis is an essential step in tumor growth and metastasis. The initiation of tumor angiogenesis is dictated by a shift in the balance between proangiogenic and antiangiogenic gene expression programs. Roquin2 is a zinc-finger RNA-binding protein with important roles in mediating the expression of inflammatory genes, such as TNF, IL6 and PTGS2, which are also important angiogenic factors. In this study, we demonstrate that Roquin2 functions as a potent tumor angiogenesis regulator that inhibits breast tumor-induced angiogenesis by selectively destabilizing mRNA of proangiogenic gene transcripts, including endoglin (ENG), endothelin-1 (EDN1), vascular endothelial growth factor B (VEGFB) and platelet derived growth factor C (PDGFC). Roquin2 recognizes and binds the stem-loop structure in the 3'untranslated region (3'UTR) of these mRNAs via its ROQ domain to destabilize mRNA. Moreover, we found that Roquin2 expression was reduced in breast cancer cells and tissues, and associated with poor prognosis in breast cancer patients. Overexpression of Roquin2 inhibited breast tumor-induced angiogenesis in vitro and in vivo, whereas silencing Roquin2 enhanced tumor angiogenesis. In vivo induction of Roquin2 by adenovirus significantly suppressed breast tumor growth, metastasis and angiogenesis. Taken together, our results identify that Roquin2 is a novel breast cancer suppressor that inhibits tumor angiogenesis by selectively downregulating the expression of proangiogenic genes.

MicroRNA-mediated vascular intercellular communication is altered in chronic kidney disease.

Chronic kidney disease (CKD) is an independent risk factor for the development of coronary artery disease (CAD). For both, CKD and CAD, the intercellular transfer of microRNAs (miRs) through extracellular vesicles (EVs) is an important factor of disease development. Whether the combination of CAD and CKD affects endothelial function through cellular crosstalk of EV-incorporated miRs is still unknown. Out of 172 screened CAD patients, 31 patients with CAD + CKD were identified and matched with 31 CAD patients without CKD. Additionally, 13 controls without CAD and CKD were included. Large EVs from CAD + CKD patients contained significantly lower levels of the vasculo-protective miR-130a-3p and miR-126-3p compared to CAD patients and controls. Flow cytometric analysis of plasma-derived EVs revealed significantly higher numbers of endothelial cell-derived EVs in CAD and CAD + CKD patients compared to controls. EVs from CAD + CKD patients impaired target human coronary artery endothelial cell (HCAEC) proliferation upon incubation in vitro. Consistent with the clinical data, treatment with the uraemia toxin indoxyl sulfate (IS)-reduced miR-130a-3p levels in HCAEC-derived EVs. EVs from IS-treated donor HCAECs-reduced proliferation and re-endothelialization in EV-recipient cells and induced an anti-angiogenic gene expression profile. In a mouse-experiment, intravenous treatment with EVs from IS-treated endothelial cells significantly impaired endothelial regeneration. On the molecular level, we found that IS leads to an up-regulation of the heterogenous nuclear ribonucleoprotein U (hnRNPU), which retains miR-130a-3p in the cell leading to reduced vesicular miR-130a-3p export and impaired EV-recipient cell proliferation. Our findings suggest that EV-miR-mediated vascular intercellular communication is altered in patients with CAD and CKD, promoting CKD-induced endothelial dysfunction.

Dual-Acting Antiangiogenic Gene Therapy Reduces Inflammation and Regresses Neovascularization in Diabetic Mouse Retina

Intravitreal injections of anti-vascular endothelial growth factor drugs have become the gold standard treatment for diabetic retinopathy (DR). However, several patients are classified as non-responders or poor responders to treatment. Therefore, it is essential to study alternative target molecules. We have previously shown that the progression of DR in the Ins2Akita mouse reflects the imbalance between pro- and anti-angiogenic molecules found in the human retina. We report, for the first time, the therapeutic potential of a dual-acting antiangiogenic non-viral gene therapy. We have used an expressing vector encoding both the pigment epithelium-derived factor gene and a short hairpin RNA (shRNA) targeted to the placental growth factor to restore the balance between these factors in the retina. Twenty-one days after a single subretinal injection, we observed a marked decrease in the inflammatory response in the neural retina and in the retinal pigment epithelium, together with reduced vascular retinal permeability in the treated diabetic mouse. These results were accompanied by the restoration of the retinal capillary network and regression of neovascularization, with significant improvement of DR hallmarks. Concomitant with the favorable therapeutic effects, this approach did not affect retinal ganglion cells. Hence our results provide evidence toward the use of this approach in DR treatment.

Prediabetes uncovers differential gene expression at fasting and in response to oral glucose load in immune cells

Metabolic disorders including diabetes are associated with immune cell dysfunction. However, the effect of normal glucose metabolism or impairment thereof on immune cell gene expression is not well known. Hence, in this cross-sectional pilot study, we sought to determine the differences in gene expression in the peripheral blood mono-nuclear cells (PBMCs) of normal glucose tolerant (NGT) and prediabetic (PD) Asian Indian men, at fasting and in response to 75g oral glucose load. Illumina HT12 bead chip-based microarray was performed on PBMCs at fasting and 2-h post load conditions for NGT (N=6) and PD (N=9) subjects. Following normalization and due quality control of the raw data, differentially expressed genes (DEGs) under different conditions within and across the two groups were identified using GeneSpring GX V12.0 software. Paired and unpaired Student's t-tests were applied along with fold change cut-offs for appropriate comparisons. Validation of the microarray data was carried out through real-time qPCR analysis. Significantly regulated biological pathways were analyzed by employing DEGs and DAVID resource. Deconvolution of the DEGs between NGT and PD subjects at fasting was performed using CIBERSORT and genes involved in regulatory T-cell (Treg) function were further analyzed for biological significance. Glucose load specifically altered the expression of 112 genes in NGT and 356 genes in PD subjects. Biological significance analysis revealed transient up-regulation of innate and adaptive immune response related genes following oral glucose load in NGT individuals, which was not observed in PD subjects. Instead, in the PD group, glucose load led to an increase in the expression of pro-atherogenic and anti-angiogenic genes. Comparison of gene expression at fasting state in PD versus NGT revealed 21,707 differentially expressed genes. Biological significance analysis of the immune function related genes between these two groups (at fasting) revealed higher gene expression of members of the TLR signaling, MHC class II molecules, and T-cell receptor, chemotaxis and adhesion pathways in PD subjects. Expression of interferon-γ (IFN-γ) and TNFα was higher and that of type-1 interferons and TGF-β was lower at fasting state in PD subjects compared to NGT. Additionally, expression of multiple proteasome subunits and protein arginine methyl transferase genes (PRMTs) were higher and that of Treg specific genes was significantly distinct at fasting in PD subjects compared to NGT. Prediabetes uncovers constitutive TLR activation, enhanced IFN-γ signaling, and Treg dysfunction at fasting along with altered gene expression response to oral glucose load.

The Anti-Breast Cancer Effects of Green-Synthesized Zinc Oxide Nanoparticles Using Carob Extracts.

The use of nanoparticles synthesized by the green method to treat cancer is fairly recent. The aim of this study was to evaluate cytotoxicity, apoptotic and anti-angiogenic effects and the expression of involved genes, of Zinc Oxide Nanoparticles (ZnO-NPs) synthesized with Carob extracts on different human breast cancer cell lines. ZnO-NPs were synthesized using the extracts of Carob and characterized with various analytical techniques. The MCF-7 and MDA-MB231 cells were treated at different times and concentrations of ZnO-NPs. The cytotoxicity, apoptosis, and anti-angiogenic effects were examined using a series of cellular assays. Expression of apoptotic genes (Bax and Bcl2) and anti-angiogenic genes, Vascular Endothelial Growth Factor (VEGF) and its Receptor (VEGF-R) in cancer cells treated with ZnO-NPs were examined with Reverse Transcriptionquantitative Polymerase Chain Reaction (RT-qPCR). The anti-oxidant activities of ZnO-NPs were evaluated by ABTS and DPPH assay. Exposure of cells to ZnO-NPs resulted in a dose-dependent loss of cell viability. The IC50 values at 24, 48, and 72 hours were 125, 62.5, and 31.2μg/ml, respectively (p<0.001). ZnO-NPs treated cells showed, in fluorescent microscopy, that ZnO-NPs are able to upregulate apoptosis and RT-qPCR revealed the upregulation of Bax (p<0.001) and downregulation of Bcl-2 (p<0.05). ZnO-NPs increased VEGF gene expression while decreasing VEGF-R (p<0.001). The anti-oxidant effects of ZnO-NPs were higher than the control group and were dose-dependent (p<0.001). ZnO-NPs synthetized using Carob extract have the ability to eliminate breast cancer cells and inhibit angiogenesis, therefore, they could be used as an anticancer agent.

Mdm2-mediated neddylation of pVHL blocks the induction of antiangiogenic factors.

Mutations in the tumor suppressor TP53 are rare in renal cell carcinomas. p53 is a key factor for inducing anti-angiogenic genes and RCC are highly vascularized, which suggests that p53 is inactive in these tumors. One regulator of p53 is the Mdm2 oncogene, which is correlated with high-grade, metastatic tumors. However, the sole activity of Mdm2 is not just to regulate p53, but it can also function independent of p53 to regulate the early stages of metastasis. Here, we report that the oncoprotein Mdm2 can bind directly to the tumor suppressor VHL, and conjugate nedd8 to VHL within a region that is important for the p53-VHL interaction. Nedd8 conjugated VHL is unable to bind to p53 thereby preventing the induction of anti-angiogenic factors. These results highlight a previously unknown oncogenic function of Mdm2 during the progression of cancer to promote angiogenesis through the regulation of VHL. Thus, the Mdm2-VHL interaction represents a pathway that impacts tumor angiogenesis.

Reduced expression of microRNA-130a promotes endothelial cell senescence and age-dependent impairment of neovascularization.

Aging is associated with impaired neovascularization in response to ischemia. MicroRNAs are small noncoding RNAs emerging as key regulators of physiological and pathological processes. Here we investigated the potential role of microRNAs in endothelial cell senescence and age-dependent impairment of neovascularization. Next generation sequencing and qRT-PCR analyses identified miR-130a as a pro-angiogenic microRNA which expression is significantly reduced in old mouse aortic endothelial cells (ECs). Transfection of young ECs with a miR-130a inhibitor leads to accelerated senescence and reduced angiogenic functions. Conversely, forced expression of miR-130a in old ECs reduces senescence and improves angiogenesis. In a mouse model of hindlimb ischemia, intramuscular injection of miR-130a mimic in older mice restores blood flow recovery and vascular densities in ischemic muscles, improves mobility and reduces tissue damage. miR-130a directly targets antiangiogenic homeobox genes MEOX2 and HOXA5. MEOX2 and HOXA5 are significantly increased in the ischemic muscles of aging mice, but forced expression of miR-130a reduces the expression of these factors. miR-130a treatment after ischemia is also associated with increased number and improved functional activities of pro-angiogenic cells (PACs). Forced expression of miR-130a could constitute a novel strategy to improve blood flow recovery and reduce ischemia in older patients with ischemic vascular diseases.

Aberrant retinoic acid production in the decidua: Implications for pre-eclampsia.

Fine-tuning of the endometrium during the evanescent 'window of implantation' relies upon an array of diverse and redundant signaling molecules, particularly the ovarian steroids E2 and P4, but also growth factors, eicosanoids, and vitamins including the vitamin A compounds (retinoids). Pregnancy complications such as preeclampsia (PE) can result from aberrations in the production or function of these molecules that arise during this critical period of decidual development. Such aberrations may be reflected by incomplete decidualization, reduced spiral artery modification, and/or loss of immune tolerance to the developing fetus. Our understanding of the role of the active retinoid metabolite all-trans retinoic acid (RA) in maintaining immune balance in certain tissues, along with data describing its role in decidualization, present a compelling argument that aberrant RA signaling in the decidua can play a significant role in the etiology of PE. Recent findings that decidualization and expression of the anti-angiogenic gene product, 'soluble fms-like tyrosine kinase-1' (sFLT1) are negatively correlated and that sFLT1 expression is directly inhibited by RA, provide additional evidence of the critical role of this retinoid in regulating early vascular development in the decidua. This review provides insight into the production and function of RA in the decidua and how modifications in its metabolism and signaling might lead to certain pregnancy disorders such as PE.

Enhancing Antitumor Efficacy of Heavily Vascularized Tumors by RAMBO Virus through Decreased Tumor Endothelial Cell Activation.

Vascularization is a common pathology for many solid tumors, and therefore anti-angiogenic strategies are being investigated as a therapeutic target for treatment. Numerous studies are also being conducted regarding the effects of oncolytic viruses, including ImlygicTM, an FDA approved oncolytic herpes simplex virus-1 (oHSV) for the treatment of highly vascularized tumors such as Kaposi sarcoma (NCT04065152), and brain tumors. To our knowledge, the effects of combining oncolytic HSV with angiogenesis inhibition on endothelial cell activation has not been previously described. Here, we tested the effects of Rapid Antiangiogenesis Mediated By Oncolytic Virus (RAMBO), an oHSV which expresses a potent anti-angiogenic gene Vasculostatin on endothelial cell activation in heavily vascularized solid tumors. oHSV treatment induces endothelial cell activation, which inhibits virus propagation and oncolysis in adjacent tumor cells in vitro. Consistently, this was also observed in intravital imaging of intracranial tumor-bearing mice in vivo where infected tumor endothelial cells could efficiently clear the virus without cell lysis. Quantitative real-time PCR (Q-PCR), leukocyte adhesion assay, and fluorescent microscopy imaging data, however, revealed that RAMBO virus significantly decreased expression of endothelial cell activation markers and leukocyte adhesion, which in turn increased virus replication and cytotoxicity in endothelial cells. In vivo RAMBO treatment of subcutaneously implanted sarcoma tumors significantly reduced tumor growth in mice bearing sarcoma compared to rHSVQ. In addition, histological analysis of RAMBO-treated tumor tissues revealed large areas of necrosis and a statistically significant reduction in microvessel density (MVD). This study provides strong preclinical evidence of the therapeutic benefit for the use of RAMBO virus as a treatment option for highly vascularized tumors.

Inhibition of mTOR via an AAV-Delivered shRNA Tested in a Rat OIR Model as a Potential Antiangiogenic Gene Therapy.

PurposeRecent studies have shown that inhibitors of the mechanistic target of rapamycin (mTOR) play important roles in proliferating endothelial cells within the retinal vasculature. Here we explore the effects of inhibiting mTOR as a potential gene therapeutic against pathological retinal angiogenesis in a rat model of oxygen-induced retinopathy (OIR).MethodsSprague-Dawley pups were used to generate the OIR model, with a recombinant adeno-associated virus expressing an shRNA (rAAV2-shmTOR-GFP) being administered via intravitreal injection on returning the rats to normoxia, with appropriate controls. Immunohistochemistry and TUNEL assays, as well as fluorescein angiography, were performed on transverse retinal sections and flat mounts, respectively, to determine the in vivo effects of mTOR inhibition.ResultsCompared with normal control rats, as well as OIR model animals that were either untreated (20.95 ± 6.85), mock-treated (14.50 ± 2.47), or injected with a control short hairpin RNA (shRNA)-containing virus vector (16.64 ± 4.92), rAAV2-shmTOR-GFP (4.28 ± 2.86, P = 0.00103) treatment resulted in dramatically reduced neovascularization as a percentage of total retinal area. These results mirrored quantifications of retinal avascular area and vessel tortuosity, with rAAV2-shmTOR-GFP exhibiting significantly greater therapeutic efficacy than the other treatments. The virus vector was additionally shown to reduce inflammatory cell infiltration into retinal tissue and possess antiapoptotic properties, both these processes having been implicated in the pathophysiology of angiogenic retinal disorders.ConclusionsTaken together, these results demonstrate the strong promise of rAAV2-shmTOR-GFP as an effective and convenient gene therapy for the treatment of neovascular retinal diseases.

Effect of human Wharton's Jelly or adipose derived mesenchymal stem cells culture supernatant on endothelial cells angiogenesis

Objective: To compare the curative effect of mesenchymal stem cells derived from human Wharton's Jelly(WJ-MSC) or adipose(AD-MSC) culture supernatant on endothelial cells angiogenesis. Methods: WJ-MSC and AD-MSC were isolated, identified, and the culture supernatant of stem cells was collected.The WJ-MSC or AD-MSC supernatant co-cultured with the endothelial cells. The expression levels of pro-angiogenic and anti-angiogenic genes of endothelial cells were assessed using qRT-PCR analysis, and the effects of stem cell culture supernatant on angiogenesis were evaluated by performing a tube formation assay in vitro. Results: After adding WJ-MSC and AD-MSC culture supernatant, the expression levels of pro-angiogenic genes in endothelial cells were upregulated, and the expression levels of anti-angiogenic genes were downregulated significantly in both experimental groups compared to the control group (P<0.01), and tube formation of endothelial cells was also significantly increased in both experimental groups as determined by the increase of the tube length ((43.2±9.2) mm vs (94.3±13.2)mm, (86.1±7.2)mm, P<0.01). Conclusion: The results showed that AD-MSC culture supernatant can promote endothelial cells angiogenesis and its curative effect is similar to that of WJ-MSC.