Pro-angiogenic Mediators Research Articles

Epigenetic regulation of Nrf2-Mediated angiogenesis in diabetic foot ulcer progression: Role of histone deacetylases

Nuclear factor E2-related factor 2 (Nrf2), a redox-sensitive transcription factor, regulates proangiogenic mediators, and antioxidant and detoxification enzymes. However, hitherto its regulation in the progression of DFU was poorly examined. The regulation of Nrf2 has been reported to be affected by various factors, including histone deacetylase (HDACs) and DNA methylation. The present study aimed to profile all classes of HDACs and correlate them with Nrf2 and angiogenic markers in the tissue biopsies of different grades of DFU patients (n = 20 in each grade). The gene expression profile of Nrf2 and its downstream targets, angiogenic markers, and all classes of HDACs were assessed using qPCR. Spearman's correlation was performed to analyze the correlation of HDACs with Nrf2 and its downstream targets along with angiogenic markers. We observed a progressive decrease in the gene expression of Nrf2 and angiogenic markers such as VEGF, HIF-1α, and SDF-1α and also an increase in the TSP-2 expression in different grades of DFU. In parallel, a significant downregulation of HDAC2/8 and SIRT1/2/4 has been observed in various grades of DFU subjects. On the other hand, HDAC1/3/4/11 and SIRT3/5/6/7 showed upregulation in different grades of DFU and the maximum increase was observed in Grade 3 patients. A significant negative correlation between Nrf2 and HDAC4, angiogenic markers, and HDAC4 suggested the pivotal role of the HDAC4-regulated Nrf2-mediated angiogenesis among DFU subjects. We have generated a first line of evidence on the epigenetic regulation of Nrf2 and its correlation with angiogenesis in the progression of diabetic foot ulcers.

Circulating vascular endothelial growth factor receptor-3, a pro-lymphangiogenic and pro-angiogenic mediator, is decreased in pre-eclampsia.

To compare circulating levels of vascular endothelial growth factor receptor 3 (VEGFR-3) in women with pregnancy-induced hypertension (PIH) and in non-pregnant (NP) and healthy pregnant (HP) women. We conducted a case-control study including PIH (n = 135), HP (n = 68), and NP (n = 49) women from southeastern Brazil. PIH were diagnosed according to international guidelines, and defined as gestational hypertension (GH, n = 61) or pre-eclampsia (n = 74). VEGFR-3 was measured in plasma using ELISA. Plasma VEGFR-3 was increased in HP (1207 pg/mL) compared with NP (133 pg/mL) women; however, PIH (729 pg/mL) patients exhibited lower levels than HP women (both p < 0.05). In addition, plasma VEGFR-3 was decreased in pre-eclampsia compared with GH (537 versus 980 pg/mL; p < 0.05). When pre-eclampsia was classified according to different clinical presentations, plasma VEGFR-3 was further decreased in the cases identified as pre-eclampsia with severe features, preterm pre-eclampsia, and pre-eclampsia accompanied by small for gestational age (all p < 0.05). Our data indicate reduced circulating VEGFR-3 levels in patients with PIH, specifically in those diagnosed with pre-eclampsia. Moreover, decreased VEGFR-3 was associated with adverse clinical outcomes in pre-eclampsia. These findings expand previous evidence of reduced VEGFR-3 expression in pre-eclampsia. Future studies should investigate whether it can be used as a predictive biomarker and/or therapeutic target for pre-eclampsia.

The combination of apatinib and antigen-specific DC-induced T cells exert antitumor effects by potently improving the immune microenvironment of osteosarcoma

ObjectiveOsteosarcoma (OS) is the most common primary bone sarcoma with a high propensity for local invasion and metastasis. Although the antitumor effect of apatinib has been well confirmed in advanced OS, the synergistic effect of apatinib and immunotherapies has not yet been elucidated. MethodsIn this study, we established tumour-bearing mice and observed tumour size with low and high doses of apatinib treatments. The expression of 17 cytokines, including vascular endothelial growth factor (VEGF), was detected by protein microarray analysis. Moreover, we designed apatinib and antigen-specific dendritic cell (DC)-T combination treatment for tumour-bearing mice. Tumour growth was detected by statistical analysis of tumour size and microvessel density (MVD) counting, the protein expression of VEGF by western blotting, the cytokines interleukin 6 (IL-6), IL-17 and interferon-gamma (IFN-γ) by enzyme-linked immunosorbent assay (ELISA), and the numbers of myeloid-derived suppressor cells (MDSCs) and tumour-infiltration macrophages (TAMs) by flow cytometry. ResultsThe results showed that apatinib efficiently suppressed tumour growth, and high-dose apatinib achieved a stronger effect. The same was true for DC-T immunotherapy. However, their combination treatment revealed a better oncolytic effect. Meanwhile, apatinib or DC-T treatment inhibited the expression of VEGF and the proangiogenic mediators IL-6 and IL-17 but increased IFN-γ production. Combination therapy further reduced/increased these effects. In addition, the combination treatment reduced MDSC but enhanced TAM-M1 ratios in the OS microenvironment. These findings indicated that apatinib and antigen-specific DC-T combination therapy was more efficient in oncolysis by regulating pro-/anti-angiogenic inducers and improving the immune state in the OS microenvironment. ConclusionThis study proved that it was feasible to employ immunotherapy with therapeutic agents in OS treatment, which may provide a new approach in addition to the combination of surgery with chemotherapy in tumour treatment.

Myeloproliferative Neoplasms Transcriptome Reveals Pro-Inflammatory Signature and Enrichment in Peripheral Blood Monocyte-Related Genes

Myeloproliferative neoplasms (MPN) are hematological diseases associated with genetic driver mutations in the JAK2, CALR, and MPL genes and exacerbated oncoinflammatory status. Analyzing public microarray data from polycythemia vera (n = 41), essential thrombocythemia (n = 21), and primary myelofibrosis (n = 9) patients’ peripheral blood by in silico approaches, we found that pro-inflammatory and monocyte-related genes were differentially expressed in MPN patients’ transcriptome. Genes related to cell activation, secretion of pro-inflammatory and pro-angiogenic mediators, activation of neutrophils and platelets, coagulation, and interferon pathway were upregulated in monocytes compared to controls. Together, our results suggest that molecular alterations in monocytes may contribute to oncoinflammation in MPN.

Management of corneal neovascularization: Current and emerging therapeutic approaches.

Corneal neovascularization (CoNV) is a sight-threatening condition affecting an estimated 1.4 million people per year, and the incidence is expected to rise. It is a complication of corneal pathological diseases such as infective keratitis, chemical burn, corneal limbal stem cell deficiency, mechanical trauma, and immunological rejection after keratoplasties. CoNV occurs due to a disequilibrium in proangiogenic and antiangiogenic mediators, involving a complex system of molecular interactions. Treatment of CoNV is challenging, and no therapy thus far has been curative. Anti-inflammatory agents such as corticosteroids are the mainstay of treatment due to their accessibility and well-studied safety profile. However, they have limited effectiveness and are unable to regress more mature neovascularization. With the advent of advanced imaging modalities and an expanding understanding of its pathogenesis, contemporary treatments targeting a wide array of molecular mechanisms and surgical options are gaining traction. This review aims to summarize evidence regarding conventional and emerging therapeutic options for CoNV.

761 - Circulating levels of vascular endothelial growth factor receptor (VEGFR)-3, a pro-lymphangiogenic and pro-angiogenic mediator, are decreased in preeclampsia

761 - Circulating levels of vascular endothelial growth factor receptor (VEGFR)-3, a pro-lymphangiogenic and pro-angiogenic mediator, are decreased in preeclampsia

Membrane-derived particles shed by PSMA-positive cells function as pro-angiogenic stimuli in tumors

Cell membrane-derived particles (Mp) are rounded membrane-enclosed particles that are shed from tumor cells. Mp are formed from tumor membranes and are capable of tumor targeting and immunotherapeutic agents because they share membrane homology with parental cells; thus, they are under consideration as a drug delivery vehicle. Prostate-specific membrane antigen (PSMA), a transmembrane glycoprotein with enzymatic functionality, is highly expressed in Mp and extracellular vesicles (EV) from prostate cancer (PCa) with poor clinical prognosis. Although PSMA expression was previously shown in EV and Mp isolated from cell lines and from the blood of patients with high-grade PCa, no pathophysiological effects have been linked to PCa-derived Mp. Here, we compared Mp from PSMA-expressing (PSMA-Mp) and PSMA-non-expressing (WT-Mp) cells side by side in vitro and in vivo. PSMA-Mp can transfer PSMA and new phenotypic characteristics to the tumor microenvironment. The consequence of PSMA transfer to cells and increased secretion of vascular endothelial growth factor-A (VEGF-A), pro-angiogenic and pro-lymphangiogenic mediators, with increased 4E binding protein 1 (4EBP-1) phosphorylation.

Pericardial Adipose Tissue Thrombospondin-1 Associates With Antiangiogenesis in Ischemic Heart Disease

Accumulation of ectopic pericardial adipose tissue has been associated with cardiovascular complications which, in part, may relate to adipose-derived factors that regulate vascular responses and angiogenesis. We sought to characterize adipose tissue microvascular angiogenic capacity in individuals undergoing elective cardiac surgeries including aortic, valvular, and coronary artery bypass grafting. Pericardial adipose tissue was collected intraoperatively and examined for angiogenic capacity. Capillary sprouting was significantly blunted (2-fold, p<0.001) in subjects with CAD (age 60±9 years, BMI 32±4 kg/m2, LDL-C 95±46 mg/dl, n=29) compared to age-, BMI-, and LDL-C matched individuals without angiographic obstructive CAD (age 59±10 years, BMI 35±9 kg/m2, LDL-C 101±40 mg/dl, n=12). For potential mechanistic insight, we performed mRNA expression analyses using quantitative RT- PCR, and observed no significant differences in pericardial fat gene expression of pro-angiogenic mediators vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor-2 (FGF-2), and angiopoietin-1 (angpt1), or anti-angiogenic factors soluble fms-like tyrosine kinase-1 (sFlt-1) and endostatin. In contrast, mRNA expression of anti-angiogenic thrombospondin-1 (TSP-1) was significantly upregulated (2-fold, p=0.008) in CAD compared to non-CAD subjects, which was confirmed by protein western-immunoblot analysis. TSP-1 gene knockdown using shRNA lentiviral delivery significantly improved angiogenic deficiency in CAD (p<0.05). In conclusion, pericardial fat in subjects with CAD may be associated with an anti-angiogenic profile linked to functional defects in vascularization capacity. Local paracrine actions of TSP-1 in adipose depots surrounding the heart may play a role in mechanisms of ischemic heart disease.

Novel mediators regulating angiogenesis in diabetic foot ulcer healing.

A non-healing diabetic foot ulcer (DFU) is a debilitating clinical problem amounting to socioeconomic and psychosocial burdens. DFUs increase morbidity due to prolonged treatment and mortality in the case of non-treatable ulcers resulting in gangrene and septicemia. The overall amputation rate of the lower extremity with DFU ranges from 3.34% to 42.83%. Wound debridement, antibiotics, applying growth factors, negative pressure wound therapy, hyperbaric oxygen therapy, topical oxygen, and skin grafts are common therapies for DFU. However, recurrence and nonhealing ulcers are still major issues. Chronicity of inflammation, hypoxic environment, poor angiogenesis, and decreased formation of the extracellular matrix (ECM) are common impediments leading to nonhealing patterns of DFUs. Angiogenesis is crucial for wound healing since proper vessel formation facilitates nutrients, oxygen, and immune cells to the ulcer tissue to help in clearing out debris and facilitate healing. However, poor angiogenesis due to decreased expression of angiogenic mediators and matrix formation results in nonhealing and ultimately amputation. Multiple proangiogenic mediators and vascular endothelial growth factor (VEGF) therapy exist to enhance angiogenesis, but the results are not satisfactory. Thus, there is a need to investigate novel pro-angiogenic mediators that can either alone or in combination enhance the angiogenesis and healing of DFUs. In this article, we critically reviewed the existing pro-angiogenic mediators followed by potentially novel factors that might play a regulatory role in promoting angiogenesis and wound healing in DFUs.

Vascular Endothelial Growth Factor as Potential Biomarker for COVID-19 Severity.

COVID-19 is characterized by immunological responses to viral replication and coherent with endothelitis, microvascular disturbance of lung vasculature and coagulopathy. Vascular Endothelial Growth Factor (VEGF) is a proangiogenic mediator regulating endothelial changes. It is induced by proinflammatory signaling and hypoxia. We sought to determine whether VEGF levels differ between SARS-CoV-2-positive patients of different disease severity and whether VEGF might be useful in risk stratification. After retrospective screening of all SARS-CoV-2-positive patients treated in Unfallkrankenhaus Berlin in 2020, we included those with documented VEGF measurement. We extracted laboratory values and clinical parameters. An exploratory data analysis was performed to detect possible relations between VEGF level and clinical disease features. We included 167 SARS-CoV-2-positive patients of which 139 suffered from COVID-19. Seventy-one of the COVID-19 patients had to be treated in the intensive care unit (ICU), those patients exhibited higher VEGF levels than those being admitted to normal wards (535 vs 279 pg/L, P < .001). APACHE-2 (Acute Physiology And Chronic Health Evaluation Score) correlated with mortality and patients with high values showed higher VEGF concentrations on admission (456 vs 875 pg/L, p = 0.006). Receiver operating characteristic analytic revealed that the occurrence of organ dysfunctions like acute respiratory distress syndrome (ARDS), shock, or acute kidney injury could be predicted by VEGF. It was significantly higher in patients who later died compared to survivors (637 vs 389 pg/mL, P = 0.041) and predicted mortality with same accuracy as established markers. In our cohort, association of VEGF above 277 pg/L on admission with risk of ARDS could be confirmed in logistic regression adjusting for possible confounding factors (odds ratio 3.1, 95% confidence interval: 1.34-7.7). Even though there are several limitations to this retrospective study it revealed that in COVID-19 patients VEGF can contribute to the prediction of necessity of ICU, mortality and the prediction of ARDS, kidney injury or shock. Its use in risk stratification and potential pathogenetic involvement should be further investigated.

326-OR: Rod Photoreceptor Contribution to Inflammation and Angiogenesis in Diabetic Retinopathy

Purpose: Rod photoreceptors (rods) are the most numerous and metabolically active retinal cells, and are susceptible to metabolic stress in response to hyperglycemia. Yet the role of rods in diabetic retinopathy (DR) is controversial. This study aims to investigate if rods exposed to high glucose can trigger inflammatory and angiogenic responses in Müller glia (MG) and retinal microvascular endothelial cells (RMEC). Methods: The streptozocin-induced (STZ) model was used at 10 weeks of diabetes. Transcriptomic shifts in rods were analyzed by RNAseq. The mediators produced by rods were analyzed by multiplex ELISA. The effect of rod-produced mediators on RMEC permeability was assessed. Angiogenic responses of RMECs treated with rod-produced mediators were evaluated by proliferation, migration, and tube formation assays. The effect of rods on proangiogenic mediator production by RMECs and MG was evaluated by western blot. Results: Diabetes induced upregulation of transcripts for tnf (3-fold, P=0.006) and il1a (2.2-fold, P=0.001) in rods. Primary rod cultures treated with 25 mM D-glucose (high glucose) produced increased levels of VEGF (2.5-fold, p&amp;lt;0.05), TNF-α (2.4-fold, p&amp;lt;0.05), and IL-6 (2-fold, p&amp;lt;0.05) compared to rods treated with 5 mM D-glucose (normal glucose), or with 25 mM L-glucose (osmotic control). The mediators produced by rods treated with high glucose elicited increased RMEC proliferation (2.5 fold, p&amp;lt;0.001), RMEC migration (2-fold, p&amp;lt;0.001), and RMEC tube lengths (1.9-fold, p&amp;lt;0.001). The rod-produced mediators induced RMEC secretion of matrix metalloproteinase 2 (MMP-2) (8-fold increase, p&amp;lt;0.001) and of MMP-9 (5-fold increase, p&amp;lt;0.001). MG treated with rod-produced mediators increased their production of VEGF (4-fold, p&amp;lt;0.001) and of MMP-9 (5-fold, p&amp;lt;0.001). Conclusion: Rods in hyperglycemic conditions produce mediators that increase RMEC permeability, proliferation, migration, and branching and induce a proangiogenic secretory shift in RMEC and MG. Disclosure I.De la huerta: None. J.M.Poloway: None. V.Reddy: None. J.S.Penn: None. Funding National Institutes of Health (K08EY032620); International Retinal Research Foundation; Knights Templar Eye Foundation

Genetically engineered neural stem cells (NSCs) therapy for neurological diseases; state-of-the-art.

Neural stem cells (NSCs) are multipotent stem cells with remarkable self-renewal potential and also unique competencies to differentiate into neurons, astrocytes, and oligodendrocytes (ODCs) and improve the cellular microenvironment. In addition, NSCs secret diversity of mediators, including neurotrophic factors (e.g., BDNF, NGF, GDNF, CNTF, and NT-3), pro-angiogenic mediators (e.g., FGF-2 and VEGF), and anti-inflammatory biomolecules. Thereby, NSCs transplantation has become a reasonable and effective treatment for various neurodegenerative disorders by their capacity to induce neurogenesis and vasculogenesis and dampen neuroinflammation and oxidative stress. Nonetheless, various drawbacks such as lower migration and survival and less differential capacity to a particular cell lineage concerning the disease pathogenesis hinder their application. Thus, genetic engineering of NSCs before transplantation is recently regarded as an innovative strategy to bypass these hurdles. Indeed, genetically modified NSCs could bring about more favored therapeutic influences post-transplantation in vivo, making them an excellent option for neurological disease therapy. This review for the first time offers a comprehensive review of the therapeutic capability of genetically modified NSCs rather than naïve NSCs in neurological disease beyond brain tumors and sheds light on the recent progress and prospect in this context.

Lipoxin A4 (LXA4) Reduces Alkali-Induced Corneal Inflammation and Neovascularization and Upregulates a Repair Transcriptome.

To investigate the anti-inflammatory and anti-angiogenic effects of the bioactive lipid mediator LXA4 on a rat model of severe corneal alkali injury. To induce a corneal alkali injury in the right eyes of anesthetized Sprague Dawley rats. They were injured with a Φ 4 mm filter paper disc soaked in 1 N NaOH placed on the center of the cornea. After injury, the rats were treated topically with LXA4 (65 ng/20 μL) or vehicle three times a day for 14 days. Corneal opacity, neovascularization (NV), and hyphema were recorded and evaluated in a blind manner. Pro-inflammatory cytokine expression and genes involved in cornel repair were assayed by RNA sequencing and capillary Western blot. Cornea cell infiltration and monocytes isolated from the blood were analyzed by immunofluorescence and by flow cytometry. Topical treatment with LXA4 for two weeks significantly reduced corneal opacity, NV, and hyphema compared to the vehicle treatment. RNA-seq and Western blot results showed that LXA4 decreased the gene and protein expression of pro-inflammatory cytokines interleukin (IL)-1β and IL-6 and pro-angiogenic mediators matrix metalloproteinase (MMP)-9 and vascular endothelial growth factor (VEGFA). It also induces genes involved in keratinization and ErbB signaling and downregulates immune pathways to stimulate wound healing. Flow cytometry and immunohistochemistry showed significantly less infiltration of neutrophils in the corneas treated with LXA4 compared to vehicle treatment. It also revealed that LXA4 treatment increases the proportion of type 2 macrophages (M2) compared to M1 in blood-isolated monocytes. LXA4 decreases corneal inflammation and NV induced by a strong alkali burn. Its mechanism of action includes inhibition of inflammatory leukocyte infiltration, reduction in cytokine release, suppression of angiogenic factors, and promotion of corneal repair gene expression and macrophage polarization in blood from alkali burn corneas. LXA4 has potential as a therapeutic candidate for severe corneal chemical injuries.

Abstract 683: Improved Myocardial Blood Flow And Arteriogenesis With Calpain Inhibition May Be Mediated By Vascular Endothelial Growth Factor And Fibroblast Growth Factor Angiogenic Pathways In A Large-animal Model Of Metabolic Syndrome And Chronic Myocardial Ischemia

Objective: Ischemic heart disease is the leading cause of death worldwide. Under pathological conditions like myocardial ischemia, endothelial calpain proteases are activated by hypoxia, leading to aberrant angiogenesis. Our group has shown calpain inhibition increases myocardial blood flow and vascular density, microvascular vasodilation, and expression of proangiogenic vascular endothelial growth factor (VEGF) and its receptors (VEGF-R1, VEGF-R2) in a porcine model of chronic myocardial ischemia and diet-induced metabolic syndrome. We aim to assess the role of downstream factors in angiogenic pathways. Methods: Yorkshire swine were fed a high cholesterol diet for four weeks, and then underwent placement of an ameroid constrictor to the left circumflex artery. After three weeks, pigs received either daily calpain inhibitor MDL28170 (0.25mg/kg, oral) for five weeks (n = 8) or no drug (n = 7), followed by tissue harvest of ischemic myocardium. Results: Treatment was associated with a significant increase in expression of proangiogenic mediators, fibroblast growth factor (FGF) receptor 1 (FGFR1, p &lt;0.001) and matrix metalloprotease-2 (MMP-2, p &lt;0.001). Cathepsins D and L have been implicated in both pro- and antiangiogenic actions. Cathepsin D was significantly increased (p = 0.004), but Cathepsin L was unchanged (p &gt;0.99). Cathepsin L proenzyme, procathepsin L, was significantly decreased (p = 0.04), while there was a strong trend toward decreased procathepsin L:cathepsin L ratio (p = 0.09). Proangiogenic Tie-2 (p = 0.28), and antiangiogenic collagen 18 (p = 0.54) and angiostatin (p = 0.24) were unchanged. Conclusions: In metabolic syndrome, calpain inhibition led to increased proangiogenic signaling in chronically ischemic myocardium, suggesting angiogenic VEGF- and FGF-mediated pathways may work in concert with improved microvascular vasodilation to explain the observed increase in vascular density and collateral-dependent coronary perfusion.

Skeletonema marinoi Extracts and Associated Carotenoid Fucoxanthin Downregulate Pro-Angiogenic Mediators on Prostate Cancer and Endothelial Cells.

The exploration of natural preventive molecules for nutraceutical and pharmaceutical use has recently increased. In this scenario, marine microorganisms represent an underestimated source of bioactive products endowed with beneficial effects on health that include anti-oxidant, anti-inflammatory, differentiating, anti-tumor, and anti-angiogenic activities. Here, we tested the potential chemopreventive and anti-angiogenic activities of an extract from the marine coastal diatom Skeletonema marinoi Sarno and Zingone (Sm) on prostate cancer (PCa) and endothelial cells. We also tested one of the main carotenoids of the diatom, the xanthophyll pigment fucoxanthin (Fuco). Fuco from the literature is a potential candidate compound involved in chemopreventive activities. Sm extract and Fuco were able to inhibit PCa cell growth and hinder vascular network formation of endothelial cells. The reduced number of cells was partially due to growth inhibition and apoptosis. We studied the molecular targets by qPCR and membrane antibody arrays. Angiogenesis and inflammation molecules were modulated. In particular, Fuco downregulated the expression of Angiopoietin 2, CXCL5, TGFβ, IL6, STAT3, MMP1, TIMP1 and TIMP2 in both prostate and endothelial cells. Our study confirmed microalgae-derived drugs as potentially relevant sources of novel nutraceuticals, providing candidates for potential dietary or dietary supplement intervention in cancer prevention approaches.

Immuno-engineered mRNA combined with cell adhesive niche for synergistic modulation of the MSC secretome

In vitro transcribed (IVT-)mRNA has entered center stage for vaccine development due to its immune co-stimulating properties. Given the widely demonstrated safety of IVT-mRNA-based vaccines, we aimed to adopt IVT-mRNA encoding VEGF for secretory phenotype modulation of therapeutic cells. However, we observed that the immunogenicity of IVT-mRNA impairs the endogenous secretion of pro-angiogenic mediators from transfected mesenchymal stromal cells, instead inducing anti-angiogenic chemokines. This inflammatory secretome modulation limits the application potential of unmodified IVT-mRNA for cell therapy manufacturing, pro-angiogenic therapy and regenerative medicine.To uncouple immunogenicity from the protein expression functionality, we immuno-engineered IVT-mRNA with different chemically modified ribonucleotides. 5-Methoxy-uridine-modification of IVT-mRNA rescued the endogenous secretome pattern of transfected cells and prolonged secretion of IVT-mRNA-encoded VEGF.We found that high secretion of IVT-mRNA-encoded protein further depends on optimized cell adhesion. Cell encapsulation in a collagen-hyaluronic acid hydrogel increased secretion of IVT-mRNA-encoded VEGF and augmented the endogenous secretion of supporting pro-angiogenic mediators, such as HGF.Integrating minimally immunogenic mRNA technology with predesigned matrix-derived cues allows for the synergistic combination of multiple dimensions of cell manipulation and opens routes for biomaterial-based delivery of mRNA-engineered cell products. Such multimodal systems could present a more biologically relevant way to therapeutically address complex multifactorial processes such as tissue ischemia, angiogenesis, and regeneration.

Epithelial membrane protein 2 (EMP2) regulates hypoxia-induced angiogenesis in the adult retinal pigment epithelial cell lines

Pathologic retinal neovascularization is a potentially blinding consequence seen in many common diseases including diabetic retinopathy, retinopathy of prematurity, and retinal vaso-occlusive diseases. This study investigates epithelial membrane protein 2 (EMP2) and its role as a possible modulator of angiogenesis in human retinal pigment epithelium (RPE) under hypoxic conditions. To study its effects, the RPE cell line ARPE-19 was genetically modified to either overexpress EMP2 or knock down its levels, and RNA sequencing and western blot analysis was performed to confirm the changes in expression at the RNA and protein level, respectively. Protein expression was evaluated under both normoxic conditions or hypoxic stress. Capillary tube formation assays with human umbilical vein endothelial cells (HUVEC) were used to evaluate functional responses. EMP2 expression was found to positively correlate with expression of pro-angiogenic factors HIF1α and VEGF at both mRNA and protein levels under hypoxic conditions. Mechanistically, EMP2 stabilized HIF1α expression through downregulation of von Hippel Lindau protein (pVHL). EMP2 mediated changes in ARPE-19 cells were also found to alter the secretion of a paracrine factor(s) in conditioned media that can regulate HUVEC migration and capillary tube formation in in vitro functional angiogenesis assays. This study identifies EMP2 as a potential mediator of angiogenesis in a human RPE cell line. EMP2 levels positively correlate with pro-angiogenic mediators HIF1α and VEGF, and mechanistically, EMP2 regulates HIF1α through downregulation of pVHL. This study supports further investigation of EMP2 as a promising novel target for therapeutic treatment of pathologic neovascularization in the retina.

Abstract 14820: Pericardial Adipose Tissue Thrombospondin-1 Associates With Anti-Angiogenesis in Ischemic Heart Disease

Defective angiogenesis contributes to mechanisms of ischemic heart disease. Accumulation of ectopic pericardial adipose tissue has been associated with cardiovascular complications which, in part, may relate to adipose tissue-derived factors. We sought to characterize adipose tissue microvascular angiogenic capacity in individuals undergoing elective cardiac surgeries including aortic, valvular, and coronary artery bypass grafting (CABG). Pericardial fat was collected intraoperatively and examined for capillary sprouting capacity using an established ex vivo Matrigel-based methodology quantified for growth over seven days. Angiogenic capacity was significantly blunted (2.5-fold, p&lt;0.001) in subjects with CAD (age 59±2 years, BMI 32±1 kg/m 2 , LDL-C 92±45 mg/dl, n=37) compared to age-, BMI-, and LDL-cholesterol matched individuals without angiographic obstructive CAD (age 56±4 years, BMI 32±1 kg/m 2 , LDL-C 110±40 mg/dl, n=14). To garner potential mechanistic insight, we performed transcriptomic analyses using quantitative RT- PCR for select angiogenic regulators. We observed no significant differences in pericardial fat gene expression for pro-angiogenic mediators vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor-2 (FGF-2), and angiopoietin-1, or anti-angiogenic factors including soluble fms-like tyrosine kinase-1 (sFlt-1) and endostatin. In contrast, mRNA expression of anti-angiogenic thrombospondin-1 (TSP1) was significantly upregulated (2-fold, p&lt;0.05) in CAD subjects compared to the non-CAD group, which was confirmed by protein western-immunoblot analysis. Plasma concentrations of TSP-1 quantified by ELISA were not significantly different between the two groups. In summary, pericardial fat in subjects with CAD may be associated with an anti-angiogenic transcriptome linked to functional defects in vascularization capacity. Local paracrine actions of TSP1 in adipose depots surrounding the heart may play a role in mechanisms of ischemic heart disease.

Clinical impact of soluble Neuropilin-1 in ovarian cancer patients and its association with its circulating ligands of the HGF/c-MET axis.

Neuropilin (NRP) is a transmembrane protein, which has been shown to be a pro-angiogenic mediator and implicated as a potential driver of cancer progression. NRP-1 up-regulation in ovarian cancer tissue predicts poor prognosis. However, the clinical relevance of the soluble form of NRP-1 (sNRP-1) as a circulating biomarker in ovarian cancer patients is unknown. sNRP-1 levels were quantified in a cohort of 88 clinically documented ovarian cancer patients by a commercially available sNRP-1 enzyme-linked immunosorbent assay (ELISA) kit (Biomedica, Vienna, Austria). Patients (81.8% with FIGOIII/IV) received primary cytoreductive surgery with the aim of macroscopic complete resection (achieved in 55.7% of patients) and the recommendation of adjuvant chemotherapy in line with national guidelines. Higher levels of sNRP-1 reflected more advanced disease (FIGO III/IV) and indicated a trend towards suboptimal surgical outcome, i.e. any residual tumor. sNRP-1 was neither related to the patients' age nor the BRCA1/2 mutational status. Patients with higher sNRP-1 levels at primary diagnosis had a significantly reduced progression-free survival (PFS) (HR = 0.541, 95%CI: 0.304 - 0.963; p = 0.037) and overall survival (OS) (HR = 0.459, 95%CI: 0.225 - 0.936; p = 0.032). Principal component analysis showed that sNRP-1 levels were unrelated to the circulating hepatocyte growth factor (HGF) and the soluble ectodomain of its receptor the tyrosine kinase mesenchymal-epithelial transition (c-MET), suggesting that there is no proportional serological concentration gradient of soluble components of the NRP-1/HGF/c-MET signaling axis. In line with the previously shown tissue-based prognostic role, we demonstrated for the first time that sNRP-1 can also act as a readily accessible, prognostic biomarker in the circulation of patients with ovarian cancer at primary diagnosis. Given its known role in angiogenesis and conferring resistance to the poly ADP-ribose polymerase (PARP) inhibitor olaparib in vitro, our results encourage more detailed investigation into sNRP-1 as a potential predictive biomarker for bevacizumab and/or PARP-inhibitor treatment.

DOT1L regulates MTDH-mediated angiogenesis in triple-negative breast cancer: intermediacy of NF-κB-HIF1α axis.

DOT1L, a specific H3K79 methyltransferase, has a tumour-promoting role in various cancers, including triple-negative breast cancer (TNBC). However, the molecular mechanism by which the deregulated DOT1L promotes cancer progression is unclear. Herein, we show that a significantly higher basal level of DOTL1 strongly correlates with MTDH, an oncogene, in clinical TNBC patient cohorts and mediates TNBC progression by enhancing MTDH-induced angiogenesis. In parallel, severe combined immunodeficiency mice-bearing MDA-MB-231 cells with MTDH-Wt or MTDHΔ7 (spliced isoform of MTDH) overexpression constructs showed enhanced blood vessel formations at the tumour site in comparison with control groups. Selective inhibition of DOT1L by EPZ004777, a specific DOT1L inhibitor, or siDOT1L, significantly impaired MTDH-induced proliferation, invasion and angiogenic markers expression in TNBC cells. ChIP assay revealed that Dot1L promotes MTDH-Wt/Δ7 transcription by increasing H3K79me3 levels on its promoter. Dot1L depletion reversed this effect. Mechanistically, DOT1L-induced MTDH caused enhanced nuclear factor kappa B (NF-κB) occupancy on the hypoxia-inducible factor1α (HIF1α) promoter and increased its transcription, leading to elevated levels of proangiogenic mediators in TNBC cells. Moreover, the condition media obtained from MDA-MB-231 cells stably expressing either MTDH-Wt or MTDHΔ7 treated with EPZ004777 or Bay-11-7082 (NF-κB inhibitor) or FM19G11 (HIF1α inhibitor) significantly inhibited MTDH-induced tube formation in human umbilical vein endothelial cells, rat aortic ring sprouting and vessel formations by chick chorioallantoic membrane assay mimicking physiological angiogenic vasculature. Collectively, our findings reveal a novel epigenetic regulation of MTDH by DOTL1, which drives angiogenesis, and that the therapeutic disruption of the DOT1L-MTDH-NF-κB-HIF1α axis may have usefulness in the management of TNBC.