Endothelial Angiogenesis Research Articles

Role of transforming growth factor-β1 pathway in angiogenesis induced by chronic stress in colorectal cancer

ABSTRACT Background Chronic stress can induce stress-related hormones; norepinephrine (NE) is considered to have the highest potential in cancer. NE can stimulate the expression of hypoxia-inducible factor-1α (HIF-1α), which is associated with vascular endothelial growth factor (VEGF) secretion and tumor angiogenesis. However, the underlying mechanisms are poorly understood. Methods Tumor-bearing mice were subjected to chronic restraint stress and treated with normal saline, human monoclonal VEGF-A neutralizing antibody bevacizumab, or β-adrenergic receptor (β-AR) antagonist (propranolol). Tumor growth and vessel density were also evaluated. Human colorectal adenocarcinoma cells were treated with NE, propranolol, or the inhibitor of transforming growth factor-β (TGF-β) receptor Type I kinase (Ly2157299) in vitro. TGF-β1 in mouse serum and cell culture supernatants was quantified using ELISA. The expression of HIF-1α was measured using Real time-PCR and western blotting. Cell migration and invasion were tested. Results Chronic restraint stress attenuated the efficacy of bevacizumab and promoted tumor growth and angiogenesis in a colorectal tumor model. Propranolol blocked this effect and inhibited TGF-β1 elevation caused by chronic restraint stress or NE. NE upregulated HIF-1α expression, which was reversed by propranolol or Ly2157299. Propranolol and Ly2157199 blocked NE-stimulated cancer cell migration and invasion. Conclusions Our results demonstrate the effect of NE on tumor angiogenesis and the critical role of TGF-β1 signaling during this process. In addition, β-AR/TGF-β1 signaling/HIF-1α/VEGF is a potential signaling pathway. This study also indicates that psychosocial stress might be a risk factor which weakens the efficacy of anti-angiogenic therapy.

Upregulation of rate-limiting enzymes in cholesterol metabolism by PKCδ mediates endothelial apoptosis in diabetic wound healing

Diabetic foot ulcer (DFU) is a prevalent complication of diabetes that poses significant challenges in terms of treatment and management. It is characterized by heightened endothelial apoptosis and impaired angiogenesis. In this study, we aimed to investigate the role of protein kinase Cδ (PKCδ) in regulating endothelial apoptosis in diabetic wounds by promoting cholesterol biosynthesis. The expression of PKCδ was increased in human umbilical vascular endothelial cells (HUVECs) cultivated in high glucose medium and skin tissue isolated from diabetic mice. High glucose-induced HUVECs apoptosis was reduced by PKCδ inhibition with siRNA or rottlerin. RNA-seq identified two enzymes, 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), as the downstream of PKCδ. PKCδ knockdown or inhibition suppressed the expression of HMGCS1 and HMGCR and lowered free cholesterol (FC) levels. Cholesterol restored high glucose-induced apoptosis in siRNA- or rottlerin-treated HUVECs. In vivo use of rosuvastatin calcium, an inhibitor of HMGCR, downregulated free cholesterol levels and accelerated the wound healing process. In conclusion, PKCδ expression in endothelial cells was activated by high glucose, which subsequently upregulates the expression of two enzymes catalyzing cholesterol biosynthesis, HMGCS1 and HMGCR. Enhanced cholesterol biosynthesis raises free cholesterol levels, promotes endothelial apoptosis, and finally delays wound healing.

Circulating cluster miR-99b/let-7e/miR-125a expression associates with endothelial function, cytokine, and chemokine levels in myocardial infarction patients

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Spanish Ministry of Science and Innovation (ISCIII) co-financed by the European Regional Development Fund (ERDF), and by the Generalitat Valenciana Introduction During vascular remodelling following a myocardial infarction, miRNAs can modulate endothelial function in a post-transcriptional manner. Our study is focused on the cluster miR-99b/let-7e/miR-125a, examining its serum expression during the acute phase of a Non-ST-segment Elevation Myocardial Infarction (NSTEMI) and its expression one year later. Purpose All the miRNA miR-99b/let-7e/miR-125a cluster is hypothesized to impact endothelial function, specifically influencing cellular adhesion, proliferation, and vasculogenesis. Furthermore, it is hypothesized an association exists between the levels of miRNAs from this cluster during the acute phase of myocardial infarction and the serum levels of inflammatory cytokines and chemokines implicated in the pathology. Methods Serum RNA was extracted from control (n=41), acute NSTEMI phase (n=46), and one-year follow-up (n=24) patients. miRNA expression was quantified by qRT-PCR and miR-484 was used as endogenous control. Endothelial function was assessed by analyzing adhesion, proliferation, and vasculogenesis in human umbilical vein endothelial cells (HUVEC) and transfected with miRNA inhibitors and mimics. Serum levels of proinflammatory cytokines (IL-1β, IL-6, TNF-α, and VEGF-A), anti-inflammatory cytokines (IL-10 and IL-13), and chemokines (MCP-3, MDC, IL-8, and MIP-1α) were determined. Results The miRNA cluster components exhibited decreased expression during the acute phase of the infarction (p< 0.05) and returned to control levels after one year of follow-up. All the cluster components were high expressed in endothelial cells, and we determined the effect of these miRNAs or their inhibition on endothelial function. let-7e mimic increased (p< 0.05) cell adhesion, as well as the formation of tubular networks (p< 0.001). miR-125a inhibitor decreased cell proliferation (p< 0.05). Neither the mimic nor the inhibitor of miR-99b had effects on this endothelial function. let-7e exhibited a negative correlation with the proinflammatory cytokine TNF-α (r= -0.19, p= 0.0386) and a positive correlation with the levels of the anti-inflammatory cytokine IL-13 (r= 0.22, p= 0.0416). miR-99b showed a positive association with the chemokine MDC (r= 0.22, p= 0.0195), while the levels of miR-125a did not correlate with any of the analysed mediators. Conclusions miR-99b/let-7e/miR-125a cluster is implicated in NSTEMI and exhibits a consistent expression pattern in patients' serum, decreasing during the acute phase and returning to control levels one year after. Each of these miRNAs, especially let-7e and miR-125a, differentially affects cellular processes such as endothelial proliferation and angiogenesis. Additionally, let-7e and miR-99b miRNAs differentially impact the inflammation generated in NSTEMI. These results suggest the involvement of miRNAs from the same cluster in various processes that are coordinately activated in inflammatory situations, such as acute coronary syndrome.

UFM1 inhibits hypoxia-induced angiogenesis via promoting proteasome degradation of HIF-1α.

Angiogenesis is crucial for blood flow recovery and ischemic tissue repair of peripheral artery disease (PAD). Exploration of new mechanisms underlying angiogenesis will shed light on the treatment of PAD. Ubiquitin-fold modifier 1 (UFM1), a newly identified ubiquitin-like molecule, has been discovered to be involved in various pathophysiological processes. However, the role of UFM1 in the pathogenesis of PAD, especially in endothelial angiogenesis remains obscure, and we aimed to clarify this issue in this study. We initially found UFM1 was significantly upregulated in gastrocnemius muscles of PAD patients and hind limb ischemia mice. And UFM1 was mainly colocalized with endothelial cells in ischemic muscle tissues. Further, elevated expression of UFM1 was observed in hypoxic endothelial cells. Subsequent genetic inhibition of UFM1 dramatically enhanced migration, invasion, adhesion, and tube formation of endothelial cells under hypoxia. Mechanistically, UFM1 reduced the stability of hypoxia-inducible factor-1α (HIF-1α) and promoted the von Hippel-Lindau-mediated K48-linked ubiquitin-proteasome degradation of HIF-1α, which in turn decreased angiogenic factor VEGFA expression and suppressed VEGFA related signaling pathway. Consistently, overexpression of UFM1 inhibited the angiogenesis of endothelial cells under hypoxic conditions, whereas overexpression of HIF-1α reversed this effect. Collectively, our data reveal that UFM1 inhibits the angiogenesis of endothelial cells under hypoxia through promoting ubiquitin-proteasome degradation of HIF-1α, suggesting UFM1 might serve as a potential therapeutic target for PAD.

Abstract PO3-24-01: Mapping inter- and intra-tumor heterogeneity in Ductal Carcinoma in situ and invasive breast cancer using integrative multi-omic profiling

Abstract Background: Molecular profiling of ductal carcinoma in situ (DCIS) has shown some prognostic utility in the clinic. However, there is still an incomplete understanding of the diversity of molecular mechanisms by which DCIS progresses to invasive breast cancer (IBC).Here, we utilize integrative multi-omic profiling of co-occurring DCIS and IBC in humans as a model for mapping the relationship between tumor mutations, global gene expression and morphological changes in DCIS and IBC. Methods: We performed targeted panel DNA sequencing (Tempus xT), whole transcriptome profiling and hyperplex immunofluorescence (Lunaphore COMET) on a cohort of 39 pairs of co-occurring DCIS and IBC lesions. We performed an integrative analysis of the above multi-modal data to uncover similar and dissimilar molecular trajectories in DCIS/IBC pairs. DAVID, KEGG and Reactome databases were utilized to analyze differential pathway activity between DCIS and IBC lesions. Results: IBC samples significantly overexpressed pathways associated with cycling cells and proliferation. For DCIS, many of the enriched pathways were related to CYP enzymes and xenobiotic metabolism. We also assessed enrichment of specific transcription factor binding sites (TFBS) with the associated differentially expressed genes. The top site enriched in IBC was a promoter regulatory element of unknown function (M120 motif KRCTCNNNNMANAGC) as well as sites regulated by heat shock transcription factor 4 (HSF4). Top TFBS enriched in DCIS included MEF2, HMEF2, AR and CEBPE. The top pathways associated with IBC using DAVID were related to extracellular matrix (ECM) organization and regulation. Interestingly, the top pathways associated with DCIS also included several pathways associated with ECM organization. On Reactome pathways diagrams for ECM formation distinct components of ECM formation and organization were enriched in IBC vs DCIS lesions, with invadopodia formation components and proteoglycans specifically enriched in IBC and integrin/laminin signaling enriched in DCIS. Among the 39 cases profiled, we observed distinct patterns of DCIS/IBC pairs with highly similar mutational profiles indicative of derivation from a shared progenitor as well as pairs with little-to-no molecular concordance that likely arose independently. Even within mutationally similar DCIS/IBC pairs there were different degrees of gene expression concordance. We subdivided mutationally similar pairs into two groups based on gene expression similarity (Euclidean Distance) and identified 115 genes that differed between these groups (q < 0.05), with the dissimilar DCIS lesions hallmarked by upregulation of a variety of pathways related to B-cell function, complement cascade and cell cycle progression. Specific mutations also were related to conserved transcriptional programs in these lesions, with MCL-1 amplified tumors showing a common gene expression signature of 357 altered genes (q < 0.05) with key differences in extracellular matrix organization and adaptive immune response pathways. Dissimilarly, tumors with CKS1B copy number alterations associated with 627 differentially expressed genes (q < 0.05) with the top targets related to endothelial function and angiogenesis. Conclusions: Integrative multi-modal analysis helps us better understand tumor progression at multiple levels of biomolecular dysregulation and shows the complex interplay among genome alterations, gene expression reprogramming and tissue morphology. Such approaches may yield future biomarker signatures that better encapsulate the diverse mechanisms by which DCIS lesions evolve. Citation Format: Henry Kaplan, Alexa Dowdell, Racheli Ben Shimoli, Angela Crabtree, Ann Berry, Fred Robinson, Christopher Carney, Carlo Bifulco, Brian Piening. Mapping inter- and intra-tumor heterogeneity in Ductal Carcinoma in situ and invasive breast cancer using integrative multi-omic profiling [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-01.

Abstract 2033: Bone Marrow-Derived Macrophage Extracellular Vesicles From Diabetic Mice Suppress Angiogenic Properties In Endothelial Cells

Introduction: Peripheral artery disease (PAD), increased in diabetes, leads to mortality and disability. Diabetes promotes a proinflammatory macrophage phenotype that suppresses repair. Macrophage-derived extracellular vesicles (EVs) have been associated with tissue damage. We hypothesize that diabetes perturbs macrophage EV cargo, thereby contributing to impaired repair responses to PAD/ischemia. Methods: Unilateral ligation of the left femoral artery (FAL) was performed to induce hind limb ischemia (HLI) in diabetic Akita (C57BL/6-Ins2 Akita /J) vs. non-diabetic (NDM, C57BL/6J) male mice. Murine BMDMs were grown in Exo-free medium. Ultracentrifugation was used to isolate EVs. Western blotting, transmission electron microscopy (TEM) and ZetaView nanoparticle tracking analysis were used to characterize EV size, number, concentration, and morphology. We profiled EV microRNA (miRNA) and protein cargos from Akita vs. NDM BMDM mice. Results: BMDMs-EV size was similar in Akita and NDM mice (diameter, 20-200nm). TEM showed cup-shaped morphology. Western blot confirmed known positive and negative control exosomal markers. Comparing Akita vs. NDM BMDM EVs, a total of 3277 proteins were identified; 821 proteins were significantly differentially expressed. Enrichment analysis highlighted pathways predominantly related to oxidative stress, insulin signaling, actin cytoskeleton, CLEAR, lipid metabolism, phagosome maturation, RNA binding and DNA replication. A total of 912 EV miRNAs were identified; 39 were significantly up-regulated and 19 were down-regulated. Preliminary analysis links a number of them to endothelial function and angiogenesis. In vitro , endothelial tube formation was impaired in human iliac artery endothelial cells treated with diabetic vs. NDM BMDM EVs. BMDM treated with diabetic vs. WT EVs displayed increased expression of inflammatory genes (TNF, IL-6,) and reduced expression of mitochondrial complex I and complex II proteins. Conclusion: We identified microRNAs and distinct proteins from diabetic EVs as potential mediators of impaired endothelial function and resolution of inflammation. Targeting macrophage EVs composition might identify therapeutic approaches to prevent and treat PAD in diabetes.

The significance of Syndecan 1, a new marker for endothelial dysfunction, in cases of fetal growth retardation.

In the current study we aimed to investigate Syndecan 1 (SDC1) levels in pregnant women diagnosed with fetal growth restriction (FGR) and the relationship between SDC1 levels and clinical and doppler parameters in FGR cases associated with endothelial dysfunction, angiogenesis and uteroplacental insufficiency METHOD OF STUDY: A total of 90 pregnant women included in the study, (45 with FGR, 45 healthy control) matched by week of gestation and maternal age. Venous blood samples were collected and plasma concentrations of SDC1 were determined by a specific immunoassay. Doppler examination was performed to evaluate the relationship between the SDC1 levels and placental blood supply. Doppler parameters; meanUtA-PI(p <.001), CPR (p=.002) and CPUR (p <.001) were different between the groups, however MCA PI, umbilical artery PI and umbilical artery S/D were not (p>.05). While gestational age at delivery, birth weight, APGAR score at 1 and 5 min were significantly lower (all, p <.001) in the study group, non-reassure fetal heart rate tracing (p=.09) and NICU admission (p=.02) were significantly higher. SDC 1 level was 2,00±1,47ng/mL and 2,34±1,12ng/mL in the FGR and control groups, respectively (p=.008). In the study group SDC 1 level was 1,69±2,00 in those with gestational age below 32weeks and 2,13±1,18 in those with gestational age above 32weeks and there was a statistically significant difference between the groups (p=.015). Plasma SDC 1 concentration of 2,1850ng/mL or less had a sensitivity of 70%, a specificity of 72%, area under the ROC curve.65 (p<.005). Low maternal plasma SDC1 level may be associated with placental insufficiency and FGR. Low levels of SDC1 may be helpful as a predictor for the development of FGR during gestation.

Exploring the diagnostic value of endothelial cell and angiogenesis-related genes in Hashimoto's thyroiditis based on transcriptomics and single cell RNA sequencing

(1) BackgroundHashimoto's thyroiditis (HT) can cause angiogenesis in the thyroid gland. However, the molecular mechanism of endothelial cells and angiogenesis related genes (ARGs) has not been extensively studied in HT. (2) MethodsThe HRA001684, GSE29315 and GSE163203 datasets were included in this study. Using single-cell analysis, weighted gene co-expression network analysis (WGCNA), functional enrichment analysis, machine learning algorithms and expression analysis for exploration. And receiver operator characteristic (ROC) curves was draw. Gene set enrichment analysis (GSEA) was utilized to investigate the biological function of the biomarkers. Meanwhile, we investigated into the relationship between biomarkers and different types of immune cells. Additionally, the expression of biomarkers in the TCGA-TC dataset was examined and the mRNA-drug interaction network was constructed. (3) ResultsWe found 14 cell subtypes were obtained in HT samples after single-cell analysis. A total of 5 biomarkers (CD52, CD74, CD79A, HLA-B and RGS1) were derived, and they had excellent diagnostic performance. Then, 27 drugs targeting biomarkers were predicted. The expression analysis showed that CD74 and HLA-B were significantly up-regulated in HT samples. (4) ConclusionIn this study, 5 biomarkers (CD52, CD74, CD79A, HLA-B and RGS1) were screened and their expressions in endothelial cells was compared to offer a new reference for the recognition and management of HT.

Diosgenin improves post-myocardial infarction cardiac function via HAND2-induced angiogenesis

While diosgenin has been demonstrated effective in various cardiovascular diseases, its specific impact on treating heart attacks remains unclear. Our research revealed that diosgenin significantly improved cardiac function in a myocardial infarction (MI) mouse model, reducing cardiac fibrosis and cell apoptosis while promoting angiogenesis. Mechanistically, diosgenin upregulated the Hand2 expression, promoting the proliferation and migration of endothelial cells under hypoxic conditions. Acting as a transcription factor, HAND2 activated the angiogenesis-related gene Aggf1. Conversely, silencing Hand2 inhibited the diosgenin-induced migration of hypoxic endothelial cells and angiogenesis. In summary, these findings provide new insights into the protective role of diosgenin in MI, validating its effect on angiogenic activity and providing a theoretical basis for clinical treatment strategies.

MiR-522-3p Attenuates Cardiac Recovery by Targeting FOXP1 to Suppress Angiogenesis.

Angiogenesis is crucial for blood supply reconstitution after myocardial infarction in patients with acute coronary syndrome (ACS). MicroRNAs are recognized as important epigenetic regulators of endothelial angiogenesis. The purpose of this study is to determine the roles of miR-522-3p in angiogenesis after myocardial infarction. The expression levels of miR-522-3p in rats' plasma and in the upper part of the ligation of the heart tissues at 28 days after myocardial infarction were significantly higher than those of the sham group. miR-522-3p mimics inhibited cell proliferations, migrations, and tube formations under hypoxic conditions in HUVECs (human umbilical vein endothelial cells), whereas miR-522-3p inhibitors did the opposite. Furthermore, studies have indicated that the inhibition of miR-522-3p by antagomir infusion promoted angiogenesis and accelerated the recovery of cardiac functions in rats with myocardial infarction.Data analysis and experimental results revealed that FOXP1 (Forkhead-box protein P1) was the target gene of miR-522-3p. Our study explored the mechanism of cardiac angiogenesis after myocardial infarction and provided a potential therapeutic approach for the treatment of ischemic heart disease in the future.

Abstract 1586: Unraveling the importance of pancreatic cancer extracellular signaling to endothelial cells within the tumor microenvironment

Abstract In many solid tumors, including pancreatic ductal adenocarcinoma (PDAC), the secretion of extracellular vesicles (EVs) has been shown to support tumor progression, chemotherapeutic resistance, and metastasis. Importantly, PDAC tumors are composed of up to 80% non-tumor cells and an extracellular matrix, yet no studies have been performed to determine which cells within PDAC tumors internalized PDAC EVs. Further, the RNA-binding protein HuR plays an important role in PDAC cells, supporting the stability and translation of transcripts that aid tumor cell stress responses. Our lab has recently found that EVs harbor distinct mRNA and phosphorylated protein cargoes from donor cells. Further, HuR knockout (KO) cells produce EVs with significantly different mRNAs and phosphorylated proteins that EVs isolated from HuR wildtype (WT) cells. Specifically, proteins relating to endothelial function and angiogenesis were significantly differentially phosphorylated. Considering the functional impact of EV cargoes on recipient cells, our study aims to identify the specific cell types in the tumor microenvironment that import PDAC EVs and investigate the role of PDAC cell HuR in this signaling process. We have generated human PDAC and mouse KrasG12D Trp53R172H driven (KPC) PDAC cell lines to express an EV reporter, PalmGRET. Utilizing these cells lines in an immunocompetent mouse model of PDAC, we found that endothelial cells are major importers of PDAC EVs. We next performed bulk RNA-sequencing on endothelial cells sorted from these tumors that have (GFP+) versus have not (GFP-) imported PDAC EVs to assess the functional impact of PDAC EVs. Subsequent studies will focus on delineating the importance of this signaling axis by ablating EV secretion in addition to assessing endothelial cell function when treated with EVs from HuR WT vs. KO cells. Collectively, our study sheds light on the preferential uptake of PDAC EVs by endothelial cells and highlights the potential role of tumor cell-intrinsic HuR in this signaling process. These findings expand our understanding of the interactions between PDAC and the tumor microenvironment, potentially paving the way for targeted therapeutic interventions in this devastating disease. Citation Format: Jennifer M. Finan, Yifei Guo, Alexandra Q. Bartlett, Kevin MacPherson, Valerie Calvert, Madeline D. Hedberg, Emanuel Petricoin, Rosalie Sears, Jonathan R. Brody. Unraveling the importance of pancreatic cancer extracellular signaling to endothelial cells within the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1586.

Association of interleukin-17A and chemokine/vascular endothelial growth factor-induced angiogenesis in newly diagnosed patients with bladder cancer

BackgroundThe human interleukin-17 (IL-17) family comprises IL-17A to IL-17 F; their receptors are IL-17RA to IL-17RE. Evidence revealed that these cytokines can have a tumor-supportive or anti-tumor impact on human malignancies. The purpose of this study was to assess the expression of CXCR2, IL-17RA, and IL-17RC genes at the mRNA level as well as tissue and serum levels of IL-17A, vascular endothelial growth factor (VEGF), and transforming growth factor β (TGF-β) in patients with bladder cancer (BC) compared to control.ResultsThis study showed that gene expression of IL-17RA, IL-17RC, and CXCR2 in the tumoral tissue of BC patients was significantly upregulated compared with normal tissue. The findings disclosed a significant difference in the serum and tissue concentrations of IL-17A, VEGF, and TGF-β between the patient and the control groups, as well as tumor and normal tissues.ConclusionThis study reveals notable dysregulation of CXCR2, IL-17RA, and IL-17RC genes, alongside changes in IL-17A, VEGF, and TGF-β levels in patients with BC than in controls. These findings indicate their possible involvement in BC development and their potential as diagnostic and therapeutic targets.

Overexpression of Wnt5a promoted the protective effect of mesenchymal stem cells on Lipopolysaccharide-induced endothelial cell injury via activating PI3K/AKT signaling pathway.

Lung endothelial barrier injury plays an important role in the pathophysiology ofacute lung injury/acute respiratory distress syndrome (ALI/ARDS). Mesenchymal stem cells (MSCs) therapy has shown promise in ARDS treatment and restoration of the impaired barrier function. It has been reported that Wnt5a shows protective effects on endothelial cells. Therefore, the study aimed to investigate whether overexpression of Wnt5a could promote the protective effects of MSCs on Lipopolysaccharide (LPS)-induced endothelial cell injury. To evaluate the protective effects of MSCs overexpressing Wnt5a, we assessed the migration, proliferation, apoptosis, and angiogenic ability of endothelial cells. We assessed the transcription of protective cellular factors using qPCR and determined the molecular mechanism using Western blot analysis. Overexpression of Wnt5a upregulated the transcription of protective cellular factors in MSCs. Co-culture of MSCWnt5a promoted endothelial migration, proliferation and angiogenesis, and inhibited endothelial cell apoptosis through the PI3K/AKT pathway. Overexpression of Wnt5a promoted the therapeutic effect of MSCs on endothelial cell injury through the PI3K/AKT signaling. Our study provides a novel approach for utilizing genetically modified MSCs in the transplantation therapy for ARDS.

SNORD45A Affects Content of HIF-1α and Promotes Endothelial Angiogenic Function.

To find out the differentially expressed small nucleolar RNAs (snoRNAs) in corneal neovascularization and their effect on angiogenesis.The rat model of corneal neovascularization induced by alkali burn was established, and the differentially expressed snoRNAs were sifted by high-throughput sequencing. Human genome homologs were screened and verified in cytopathological models. Polymerase chain reactions (PCRs) and Western blot assays were applied to detect mRNA and corresponding proteins affected by the differentially expressed snoRNA. In vitro, experiments were promoted to identify whether snoRNA affects endothelial cell migration and angiogenesis.Forty-seven differentially expressed snoRNAs were sifted from transparent cornea and neovascularization. According to sequencing and cytopathological model results, SNORD45A was selected for subsequent experiments. At mRNA and protein levels, SNORD45A affected the expression of HIF-1α. SNORD45A promoted endothelial angiogenesis through endothelial cell migration and tube formation regulation.The research suggested that SNORD45A partakes in the corneal neovascularization formation and can become one of the targets for corneal neovascularization therapy.

The host transcriptional response to superinfection by influenza A virus and Streptococcus pneumoniae.

Secondary bacterial infections are the most frequent complications during influenza A virus (IAV) pandemic outbreaks, contributing to excessive morbidity and mortality in the human population. Most IAV-related deaths are attributed to Streptococcus pneumoniae (SP) infections, which usually begin within the first week of IAV infection in the respiratory tracts. Here, we focused on longitudinal transcriptional responses during a superinfection model consisting of an SP infection that follows an initial IAV infection, comparing superinfection to an IAV-only infection, an SP-only infection, and control treatments. Our longitudinal data allowed a fine analysis of gene expression changes during superinfection. For instance, we found that superinfected mice exhibited rapid gene expression induction or reduction within the first 12 h after encountering the second pathogen. Cell proliferation and immune response activation processes were upregulated, while endothelial processes, vasculogenesis, and angiogenesis were downregulated, providing promising targets for future therapeutic interventions. We further analyzed the longitudinal transcriptional responses in the context of a previously defined spectrum of the host's resistance state, revealing superinfection-specific reprogramming of resistance states, such as reprogramming of fatty acid metabolism and interferon signaling. The reprogrammed functions are compelling new targets for switching the pathogenic superinfection state into a single-infection state.

(064) Argonaute 2 Restores Erectile Function by Enhancing Angiogenesis and Reducing Reactive Oxygen Species Production in Streptozotocin (STZ)-Induced Type-1 Diabetic Mice

Abstract Introduction Diabetes mellitus (DM), a primary cause of erectile dysfunction (ED), is a pancreatic β-cell dysfunction that leads to insulin deficiency and resistance, resulting in microvascular complications. Argonaute 2 (Ago2), a catalytic engine in mammalian RNA interference, is involved in neurovascular regeneration under inflammatory conditions. Objective In the present study, we aimed to determine whether an exogenous injection of Ago2 proteins can improve erectile function in STZ-induced type-1 diabetic mice and explore the specific mechanisms of Ago2 in the process. Methods Eight-week-old male C57BL/6 mice were used for in vivo study. The detailed mechanisms were evaluated in age-matched control or STZ-induced type-1 diabetic mice. Mouse cavernous endothelial cells and human umbilical vein endothelial cells were used for in vitro study. The function of Ago2 in high-glucose condition was evaluated by tube formation assay, migration assay, immunofluorescence stain and western blot experiments. Results We report that Ago2 administration can effectively improve penile erection by enhancing cavernous endothelial cell angiogenesis and survival under diabetic conditions. We found that although Ago2 is highly expressed around blood vessels and nerves, it is significantly reduced in the penis tissue of diabetic mice. Exogenous administration of the Ago2 protein restored erectile function in diabetic mice by reducing reactive oxygen species production-signaling pathways (inducing eNOS Ser1177/NF-κB Ser536 signaling) and improving cavernous endothelial angiogenesis, migration, and cell survival. Our study provides new evidence that Ago2 mediation may be a promising therapeutic strategy and a new approach for diabetic ED treatment. Conclusions Our study provides new insights into Ago2 function and may represent a new therapeutic approach for ED and other vascular and neurological diseases. Disclosure No.

Promoting collateral formation in type 2 diabetes mellitus using ultra-small nanodots with autophagy activation and ROS scavenging

BackgroundImpaired collateral formation is a major factor contributing to poor prognosis in type 2 diabetes mellitus (T2DM) patients with atherosclerotic cardiovascular disease. However, the current pharmacological treatments for improving collateral formation remain unsatisfactory. The induction of endothelial autophagy and the elimination of reactive oxygen species (ROS) represent potential therapeutic targets for enhancing endothelial angiogenesis and facilitating collateral formation. This study investigates the potential of molybdenum disulfide nanodots (MoS2 NDs) for enhancing collateral formation and improving prognosis.ResultsOur study shows that MoS2 NDs significantly enhance collateral formation in ischemic tissues of diabetic mice, improving effective blood resupply. Additionally, MoS2 NDs boost the proliferation, migration, and tube formation of endothelial cells under high glucose/hypoxia conditions in vitro. Mechanistically, the beneficial effects of MoS2 NDs on collateral formation not only depend on their known scavenging properties of ROS (H2O2, •O2-, and •OH) but also primarily involve a molecular pathway, cAMP/PKA-NR4A2, which promotes autophagy and contributes to mitigating damage in diabetic endothelial cells.ConclusionsOverall, this study investigated the specific mechanism by which MoS2 NDs mediated autophagy activation and highlighted the synergy between autophagy activation and antioxidation, thus suggesting that an economic and biocompatible nano-agent with dual therapeutic functions is highly preferable for promoting collateral formation in a diabetic context, thus, highlighting their therapeutic potential.Graphical

Peptidylarginine Deiminases Inhibitors Decrease Endothelial Cells Angiogenic Potential by Affecting Akt Signaling and the Expression and Secretion of Angiogenic Factors.

Endothelial cells (ECs) play a crucial role in various physiological processes, particularly those related to the cardiovascular system, but also those affecting the entire organism. The biology of ECs is regulated by multiple biochemical stimuli and epigenetic drivers that govern gene expression. We investigated the angiogenic potential of ECs from a protein citrullination perspective, regulated by peptidyl-arginine deiminases (PADs) that modify histone and non-histone proteins. Although the involvement of PADs has been demonstrated in several physiological processes, inflammation-related disorders and cancer, their role in angiogenesis remains unclear. To elucidate the role of PADs in endothelial angiogenesis, we used two human EC models: primary vein (HUVECs) and microvascular endothelial cells (HMEC-1). PADs activity was inhibited using irreversible inhibitors: BB-Cl-amidine, Cl-amidine and F-amidine. We analyzed all three steps of angiogenesis in vitro : proliferation, migration, and capillary-like tube formation, as well as secretory activities, gene expression and signaling in ECs. All used PAD inhibitors reduced the histone H3 citrullination (H3cit) mark, inhibited endothelial cell migration and capillary-like tube formation, and favored an angiostatic activity in HMEC-1 cells, by increasing PEDF (pigment epithelium-derived factor) and reducing VEGF (vascular endothelial growth factor) mRNA expression and protein secretion. Additionally, BB-Cl-amidine reduced the total activity of MMPs (Matrix metalloproteinases). The observed effects were underlined by the inhibition of Akt phosphorylation.>. Our findings suggest that pharmacological inhibitors of citrullination are promising therapeutic agents to target angiogenesis.

Isoimperatorin Inhibits Angiogenesis by Suppressing VEGFR2 Signaling Pathway.

Angiogenesis involves in many pathological processes, including tumor metastasis, diabetic retinopathy, and rheumatoid arthritis. Therefore, identifying therapeutic drugs that target angiogenesis may be a promising strategy for disease treatment. Isoimperatorin is a furanocoumarin with anti-inflammatory and anti-microbial effects. However, the impacts of isoimperatorin on angiogenesis and its underlying mechanisms remain unclear. This study aimed to verify its effects on vascular endothelial growth factor (VEGF)-induced endothelial angiogenesis. We employed various assays including 5-ethynyl-2'-deoxyuridine incorporation assay, transwell migration assay, wound healing assay, tube formation assay, and Western blot to evaluate the effects of isoimperatorin on angiogenesis in vitro. Additionally, we utilized Western blot and immunofluorescence analysis to examine the activation of vascular endothelial growth factor receptor (VEGFR) 2 and its downstream signaling pathways following isoimperatorin treatment. To further validate the anti-angiogenic effects of isoimperatorin in vivo, we conducted a matrigel plug assay and established an orthotopic tumor model. We demonstrated that pretreatment with isoimperatorin inhibited VEGF-induced endothelial cell proliferation, migration, and tube formation. Isoimperatorin also suppressed angiogenesis in vivo in a matrigel plug assay and in an orthotopic tumor model. Our results revealed that isoimperatorin exhibited anti-angiogenic effects via inhibiting VEGFR2 and its downstream signaling pathways activation. Our study showed that isoimperatorin suppressed angiogenesis by targeting the VEGFR2 signaling pathway and could be a potential therapeutic agent for targeting angiogenesis.

Osteopontin Promotes Angiogenesis in the Spinal Cord and Exerts a Protective Role Against Motor Function Impairment and Neuropathic Pain After Spinal Cord Injury.

Basic science study using a hemisection spinal cord injury (SCI) model. We sought to assess the effect of blocking osteopontin (OPN) upregulation on motor function recovery and pain behavior after SCI and to further investigate the possible downstream target of OPN in the injured spinal cord. OPN is a noncollagenous extracellular matrix protein widely expressed across different tissues. Its expression substantially increases following SCI. A previous study suggested that this protein might contribute to locomotor function recovery after SCI. However, its neuroprotective potential was not fully explored, nor were the underlying mechanisms. We constructed a SCI mouse model and analyzed the expression of OPN at different time points and the particular cell distribution in the injured spinal cord. Then, we blocked OPN upregulation with lentivirus-delivering siRNA targeting OPN specifically and examined its effect on motor function impairment and neuropathic pain after SCI. The underlying mechanisms were explored in the OPN-knockdown mice model and cultured vascular endothelial cells. The proteome study revealed that OPN was the most dramatically increased protein following SCI. OPN in the spinal cord was significantly increased three weeks after SCI. Suppressing OPN upregulation through siRNA exacerbated motor function impairment and neuropathic pain. In addition, SCI resulted in an increase in vascular endothelial growth factor (VEGF), AKT phosphorylation, and angiogenesis within the spinal cord, all of which were curbed by OPN reduction. Similarly, OPN knockdown suppressed VEGF expression, AKT phosphorylation, cell migration, invasion, and angiogenesis in cultured vascular endothelial cells. OPN demonstrates a protective influence against motor function impairment and neuropathic pain following SCI. This phenomenon may result from the proangiogenetic effect of OPN, possibly due to activation of the VEGF and/or AKT pathways.