Pro-angiogenic Genes Research Articles

Differential Effects of Four Canonical Notch-Activating Ligands on c-Kit+ Cardiac Progenitor Cells.

Notch signaling, an important signaling pathway in cardiac development, has been shown to mediate the reparative functions of c-kit+ progenitor cells (CPCs). However, it is unclear how each of the four canonical Notch-activating ligands affects intracellular processes in c-kit+ cells when used as an external stimulus. Neonatal c-kit+ CPCs were stimulated using four different chimeric Notch-activating ligands tethered to Dynabeads, and the resulting changes were assessed using TaqMan gene expression arrays, with subsequent analysis by principal component analysis (PCA). Additionally, functional outcomes were measured using an endothelial cell tube formation assay and MSC migration assay to assess the paracrine capacity to stimulate new vessel formation and recruit other reparative cell types to the site of injury. Gene expression data showed that stimulation with Jagged-1 is associated with the greatest pro-angiogenic gene response, including the expression of VEGF and basement membrane proteins, while the other canonical ligands, Jagged-2, Dll-1, and Dll-4, are more associated with regulatory and epigenetic changes. The functional assay showed differential responses to the four ligands in terms of angiogenesis, while none of the ligands produced a robust change in migration. These data demonstrate how the four Notch-activating ligands differentially regulate CPC gene expression and function.

Noncaloric monosaccharides induce excessive sprouting angiogenesis in zebrafish via foxo1a-marcksl1a signal.

Artificially sweetened beverages containing noncaloric monosaccharides were suggested as healthier alternatives to sugar-sweetened beverages. Nevertheless, the potential detrimental effects of these noncaloric monosaccharides on blood vessel function remain inadequately understood. We have established a zebrafish model that exhibits significant excessive angiogenesis induced by high glucose, resembling the hyperangiogenic characteristics observed in proliferative diabetic retinopathy (PDR). Utilizing this model, we observed that glucose and noncaloric monosaccharides could induce excessive formation of blood vessels, especially intersegmental vessels (ISVs). The excessively branched vessels were observed to be formed by ectopic activation of quiescent endothelial cells (ECs) into tip cells. Single-cell transcriptomic sequencing analysis of the ECs in the embryos exposed to high glucose revealed an augmented ratio of capillary ECs, proliferating ECs, and a series of upregulated proangiogenic genes. Further analysis and experiments validated that reduced foxo1a mediated the excessive angiogenesis induced by monosaccharides via upregulating the expression of marcksl1a. This study has provided new evidence showing the negative effects of noncaloric monosaccharides on the vascular system and the underlying mechanisms.

A thermosensitive hydrogel for the sustained delivery of exosomes extracted from menstrual blood mesenchymal stem cells and frizzled antibody on triple-negative breast cancer cells in vitro

Due to the heterogeneity of triple-negative breast cancer (TNBC), conventional treatments usually respond poorly and effective targeted therapies for this type of cancer are rare. Wnt signaling plays an important role in breast cancer metastasis and proliferation, as shown by analyzes of gene expression profiling and genome-wide sequencing. In addition, the role of endometrial cells in regulating angiogenesis during the menstrual cycle as an angiostatic state associated with the end of the cycle has been well established. Therefore, exosomes of menstrual blood-derived stem cells (Exo-Mens) could play a role as effective inhibitors of tumor-induced angiogenesis. However, repeated administration and systemic toxicity are major problems with conventional drug delivery methods. To minimize these drawbacks and maximize the therapeutic benefits of the drugs, hydrogels provide convenient methods for drug delivery. The aim of this study was therefore to investigate the effects of hydrogels containing Exo-Mens and Frizzled antibodies (anti-FZ) on breast cancer cells. First, the optimization and characterization of the hydrogel were performed, then the cell lines (MDA-MB-231 and HUVEC) were studied in four groups: Hydrogel (control), Hydrogel with anti-FZ, Exo-Mens and FZ + Exo. First, the IC50 of anti-FZ and Exo-Mens was determined and the degradability and release pattern of the investigated groups were measured. Western blot (VEGF, HIF-1α) and qRT-PCR (VEGF, HIF-α and KDR) were performed for the HUVEC cell line and scratch tests, colony formation and apoptosis for the MDA-MB-231 cell line. In addition, invasion and migration were performed for both cell lines. Our results showed that the optimal hydrogel is a suitable option for sustained release of the studied groups. The loaded compounds could be released within 7 days. The angiogenic activity of dual anti-FZ and Exo-Mens within the hydrogel, showed a significant shift in the expression of pro-angiogenic genes and related proteins, including HIF-1α, VEGF-A and KDR, in the treated HUVECs. The hydrogel treatment groups were also able to reduce migration and invasion in both cell lines compared to the control group. In addition, a reduction in colony formation and an increase in apoptosis were observed, especially in the cancer cells exposed to anti-FZ. Angiogenesis as well as proliferation and invasion of tumor cells were inhibited by the developed hydrogel system containing anti-FZ, Exo-Mens and a combination of both with slow release.

A point-of-research decision in synovial tissue engineering: Mesenchymal stromal cells, tissue derived fibroblast or CTGF-mediated mesenchymal-to-fibroblast transition

Rheumatoid arthritis (RA) and osteoarthritis (OA) are prevalent inflammatory joint diseases characterized by synovitis, cartilage, and bone destruction. Fibroblast-like synoviocytes (FLSs) of the synovial membrane are a decisive factor in arthritis, making them a target for future therapies. Developing novel strategies targeting FLSs requires advanced in vitro joint models that accurately replicate non-diseased joint tissue. This study aims to identify a cell source reflecting physiological synovial fibroblasts. Therefore, we newly compared the phenotype and metabolism of “healthy” knee-derived FLSs from patients with ligament injuries (trauma-FLSs) to mesenchymal stromal cells (MSCs), their native precursors. We differentiated MSCs into fibroblasts using connective tissue growth factor (CTGF) and compared selected protein and gene expression patterns to those obtained from trauma-FLSs and OA-FLSs. Based on these findings, we explored the potential of an MSC-derived synovial tissue model to simulate a chronic inflammatory response akin to that seen in arthritis. We have identified MSCs as a suitable cell source for synovial tissue engineering because, despite metabolic differences, they closely resemble human trauma-derived FLSs. CTGF-mediated differentiation of MSCs increased HAS2 expression, essential for hyaluronan synthesis. It showed protein expression patterns akin to OA-FLSs, including markers of ECM components and fibrosis, and enzymes leading to a shift in metabolism towards increased fatty acid oxidation. In general, cytokine stimulation of MSCs in a synovial tissue model induced pro-inflammatory and pro-angiogenic gene expression, hyperproliferation, and increased glucose consumption, reflecting cellular response in human arthritis. We conclude that MSCs can serve as a proxy to study physiological synovial processes and inflammatory responses. In addition, CTGF-mediated mesenchymal-to-fibroblast transition resembles OA-FLSs. Thus, we emphasize MSCs as a valuable cell source for tools in preclinical drug screening and their application in tissue engineering.

Epigenetics and Control of Tumor Angiogenesis in Melanoma: An Update with Therapeutic Implications.

Angiogenesis, the formation of new blood vessels from pre-existing ones, is a crucial process in the progression and metastasis of melanoma. Recent research has highlighted the significant role of epigenetic modifications in regulating angiogenesis. This review comprehensively examines the current understanding of how epigenetic mechanisms, including DNA methylation, histone modifications, and non-coding RNAs, influence angiogenic pathways in melanoma. DNA methylation, a key epigenetic modification, can silence angiogenesis inhibitors such as thrombospondin-1 and TIMP3 while promoting pro-angiogenic factors like vascular endothelial growth factor (VEGF). Histone modifications, including methylation and acetylation, also play a pivotal role in regulating the expression of angiogenesis-related genes. For instance, the acetylation of histones H3 and H4 is associated with the upregulation of pro-angiogenic genes, whereas histone methylation patterns can either enhance or repress angiogenic signals, depending on the specific histone mark and context. Non-coding RNAs, particularly microRNAs (miRNAs) further modulate angiogenesis. miRNAs, such as miR-210, have been identified as key regulators, with miR-9 promoting angiogenesis by targeting E-cadherin and enhancing the expression of VEGF. This review also discusses the therapeutic potential of targeting epigenetic modifications to inhibit angiogenesis in melanoma. Epigenetic drugs, such as DNA methyltransferase inhibitors (e.g., 5-azacytidine) and histone deacetylase inhibitors (e.g., Vorinostat), have shown promise in preclinical models by reactivating angiogenesis inhibitors and downregulating pro-angiogenic factors. Moreover, the modulation of miRNAs and lncRNAs presents a novel approach for anti-angiogenic therapy.

Abstract Tu046: Mechanisms of Epicardium-Directed Cardiac Repair

Background: In the adult heart, the epicardium becomes activated by myocardial infarction (MI), contributing to cardiac remodeling primarily through the secretion of paracrine factors. However, there is a limited understanding of the cellular and molecular mechanisms that govern epicardial function and promote pro-reparative signaling pathways. Thus, enhancing or restoring the pro-regenerative characteristics of the epicardium may benefit cardiac repair after MI. We hypothesize that epicardial-derived cells preserve cardiac function during ischemic injury through the limited secretion of pro-reparative paracrine factors, such as slit guidance ligand 2 (Slit2), which enhances angiogenesis and vascular stability. Methods: We used the Wt1 (Wilms Tumor 1) CreERT2 ; R26 tdTomato mouse line for tamoxifen-inducible epicardial-specific lineage tracing and Pdgfra nGFP to label cardiac fibroblasts ( Wt1 CreERT2 ; R26 tdT ; Pdgfra nGFP ). Tagged epicardial-derived cells (tdTomato + ) from sham and 7-day post-MI mice were isolated by fluorescence-activated cell sorting and sequenced by single-cell RNA sequencing. To delineate Slit2-directed cell and molecular mechanisms, we performed in vitro studies and utilized adeno-associated viral vectors for gene therapy in a mouse model of MI. Results: We identified 9 transcriptionally distinct epicardial subpopulations contributing to paracrine signaling induced by MI. We found enriched expression of various chemokines in epicardial-derived stromal cells, while genes associated with Wnt signaling and epithelial-to-mesenchymal transition (EMT) were highly expressed in Wt1 -expressing clusters. Isolated cardiac fibroblasts treated with an adenoviral vector expressing Slit2 exhibited no differences in myofibroblast formation or collagen production following treatment with activation stimuli (TGFβ1/Ang II) but showed significant increases in the expression of pro-angiogenic gene programs. Cardiac-targeted AAV9-Slit2 overexpression in mice subjected to MI led to a striking increase in cardiac ejection fraction, expression of angiogenic-modulating genes, vessel density, and endothelial cell proliferation but did not alter fibrotic deposition. Conclusions: Our study reveals the dynamic role of epicardial cells during the post-MI cardiac repair process and delineates a Slit2-Robo dependent mechanism governing cardiac fibroblast and endothelial cell crosstalk to modulate angiogenesis.

Apoptotic Extracellular Vesicles from Supernumerary Tooth-Derived Pulp Stem Cells Transfer COL1A1 to Promote Angiogenesis via PI3K/Akt/VEGF Pathway.

Angiogenesis is a tightly controlled process that initiates the formation of new vessels and its dysfunction can lead to life-threatening diseases. Apoptotic extracellular vesicles (ApoEVs) have emerged as a proangiogenic agent with high safety and isolation efficiency profile, and ApoEVs from supernumerary tooth-derived pulp stem cells (SNTSC-ApoEVs) have their unique advantages with an easily accessible parental cell source and non-invasive cell harvesting. However, the detailed characteristics of SNTSC-ApoEVs are largely unknown. This study aimed to investigate the proangiogenic capacity and function molecule of SNTSC-ApoEVs. SNTSC-ApoEVs were isolated and characterized. In vitro effects of SNTSC-ApoEVs on the proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) were evaluated by CCK-8, wound healing, transwell, and tube formation assays. The mRNA and protein levels of proangiogenic genes were quantified by qRT-PCR, Western blot, and immunofluorescence analysis. A Matrigel plug model was established in 6-week-old male nu/nu mice for one week, and the in vivo impact of SNTSC-ApoEVs on micro-vessel formation was assessed by histological analysis. Proteomic analysis and RNA sequencing were performed to explore the active ingredients and underlying mechanisms. SNTSC-ApoEVs enhanced the proliferation, migration, and angiogenesis of HUVECs in vitro. In the Matrigel plug model in vivo, SNTSC-ApoEVs promoted CD31-positive luminal structure formation. Apart from expressing general ApoEV markers, SNTSC-ApoEVs were enriched with multiple proteins related to extracellular matrix-cell interactions. Mechanistically, SNTSC-ApoEVs transferred COL1A1 to HUVECs and promoted endothelial functions by activating the PI3K/Akt/VEGF cascade. SNTSC-ApoEVs can promote angiogenesis by transferring the functional molecule COL1A1 and activating the PI3K/Akt/VEGF pathway, making SNTSC-ApoEVs a promising strategy for the treatment of angiogenesis-related diseases.

Mobilizing Endogenous Progenitor Cells Using pSDF1α-Activated Scaffolds Accelerates Angiogenesis and Bone Repair in Critical-Sized Bone Defects.

Mobilizing endogenous progenitor cells to repair damaged tissue in situ has the potential to revolutionize the field of regenerative medicine, while the early establishment of a vascular network will ensure survival of newly generated tissue. In this study, a gene-activated scaffold containing a stromal derived factor 1α plasmid (pSDF1α), a pro-angiogenic gene that is also thought to be involved in the recruitment of mesenchymal stromal cells (MSCs) to sites of injury is described. It is shown that over-expression of SDF1α protein enhanced MSC recruitment and induced vessel-like structure formation by endothelial cells in vitro. When implanted subcutaneously, transcriptomic analysis reveals that endogenous MSCs are recruited and significant angiogenesis is stimulated. Just 1-week after implantation into a calvarial critical-sized bone defect, pSDF1α-activated scaffolds are recruited MSCs and rapidly activate angiogenic and osteogenic programs, upregulating Runx2, Dlx5, and Sp7. At the same time-point, pVEGF-activated scaffolds are recruited a variety of cell types, activating endochondral ossification. The early response induced by both scaffolds leads to complete bridging of the critical-sized bone defects within 4-weeks. The versatile cell-free gene-activated scaffold described in this study is capable of harnessing and enhancing the body's own regenerative capacity and has immense potential in a myriad of applications.

The impact of hypoxia preconditioning on mesenchymal stem cells performance in hypertensive kidney disease

BackgroundAutologous mesenchymal stem cells (MSCs) have emerged as a therapeutic option for many diseases. Hypertensive kidney disease (HKD) might impair MSCs’ reparative ability by altering the biomolecular properties, but the characteristics of this impairment are unclear. In our previous pre-clinical studies, we found hypoxic preconditioning (HPC) enhanced angiogenesis and suppressed senescence gene expression. Thus, we hypothesize that HPC would improve human MSCs by enhancing their functionality and angiogenesis, creating an anti-inflammatory and anti-senescence environment.MethodsMSC samples (n = 12 each) were collected from the abdominal fat of healthy kidney donors (HC), hypertensive patients (HTN), and patients with hypertensive kidney disease (HKD). MSCs were harvested and cultured in Normoxic (20% O2) or Hypoxic (1% O2) conditions. MSC functionality was measured by proliferation assays and cytokine released in conditioned media. Senescence was evaluated by senescence-associated beta-galactosidase (SA-beta-gal) activity. Additionally, transcriptome analysis using RNA-sequencing and quantitative PCR (qPCR) were performed.ResultsAt baseline, normoxic HTN-MSCs had higher proliferation capacity compared to HC. However, HPC augmented proliferation in HC. HPC did not affect the release of pro-angiogenic protein VEGF, but increased EGF in HC-MSC, and decreased HGF in HC and HKD MSCs. Under HPC, SA-β-gal activity tended to decrease, particularly in HC group. HPC upregulated mostly the pro-angiogenic and inflammatory genes in HC and HKD and a few senescence genes in HKD.ConclusionsHPC has a more favorable functional effect on HC- than on HKD-MSC, reflected in increased proliferation and EGF release, and modest decrease in senescence, whereas it has little effect on HTN or HKD MSCs.

Xenohormetic Phytochemicals Inhibit Neovascularization in Microphysiological Models of Vasculogenesis and Tumor Angiogenesis.

Xenohormesis proposes that phytochemicals produced to combat stressors in the host plant exert biochemical effects in animal cells lacking cognate receptors. Xenohormetic phytochemicals such as flavonoids and phytoalexins modulate a range of human cell signaling mechanisms but functional correlations with human pathophysiology are lacking. Here, potent inhibitory effects of grapefruit-derived Naringenin (Nar) and soybean-derived Glyceollins (Gly) in human microphysiological models of bulk tissue vasculogenesis and tumor angiogenesis are reported. Despite this interference of vascular morphogenesis, Nar and Gly are not cytotoxic to endothelial cells and do not prevent cell cycle entry. The anti-vasculogenic effects of Glyceollin are significantly more potent in sex-matched female (XX) models. Nar and Gly do not decrease viability or expression of proangiogenic genes in triple negative breast cancer (TNBC) cell spheroids, suggesting that inhibition of sprouting angiogenesis by Nar and Gly in a MPS model of the (TNBC) microenvironment are mediated via direct effects in endothelial cells. The study supports further research of Naringenin and Glyceollin as health-promoting agents with special attention to mechanisms of action in vascular endothelial cells and the role of biological sex, which can improve the understanding of dietary nutrition and the pharmacology of phytochemical preparations.

Hypoxia stress alters gene expression in the gills and spleen of greater amberjack (Serioladumerili)

Greater amberjack (Seriola dumerili) is a fish species that has significant economic and cultural value. It has a large size and grows rapidly. However, the intolerance to hypoxia poses a major obstacle to the growth of its aquaculture industry. This study focuses on the gills and spleen, two organs closely associated with the response to acute hypoxic stress. By simulating the acute hypoxic environment and using Illumina RNA-Seq technology, we explored the gills and spleen transcriptome changes in the acute hypoxia intolerant and tolerant groups of greater amberjack. It was discovered that gill tissues in the tolerant group may maintain a stable intracellular energy supply by promoting glycolysis and β-oxidation compared to the intolerant group. Additionally, it promotes angiogenesis, enhances the ability to absorb dissolved oxygen, and accelerates oxygen transport to the mitochondria, adapting to the hypoxic environment. Anti-apoptotic genes were up-regulated in gill tissues in the tolerant group compared to the intolerant group, thereby minimizing the damage of acute hypoxia. On the other hand, the spleen inhibited the TCA and energy-consuming lipid synthesis pathways to supply energy under acute hypoxic stress. Pro-angiogenic genes were down-regulated in the spleen of individuals in the tolerant group compared to the intolerant group, which may be related to organ function. The suppressed reactive oxygen species (ROS) production and the impaired immune response function of the spleen were also found. The study explored the acute hypoxic stress response in greater amberjack and the molecular mechanisms underlying its tolerance to acute hypoxia.

Multiplexed Imaging Mass Cytometry Analysis Characterizes the Vascular Niche in Pancreatic Cancer.

Oncogenesis and progression of pancreatic ductal adenocarcinoma (PDAC) are driven by complex interactions between the neoplastic component and the tumor microenvironment, which includes immune, stromal, and parenchymal cells. In particular, most PDACs are characterized by a hypovascular and hypoxic environment that alters tumor cell behavior and limits the efficacy of chemotherapy and immunotherapy. Characterization of the spatial features of the vascular niche could advance our understanding of inter- and intratumoral heterogeneity in PDAC. In this study, we investigated the vascular microenvironment of PDAC by applying imaging mass cytometry using a 26-antibody panel on 35 regions of interest across 9 patients, capturing more than 140,000 single cells. The approach distinguished major cell types, including multiple populations of lymphoid and myeloid cells, endocrine cells, ductal cells, stromal cells, and endothelial cells. Evaluation of cellular neighborhoods identified 10 distinct spatial domains, including multiple immune and tumor-enriched environments as well as the vascular niche. Focused analysis revealed differential interactions between immune populations and the vasculature and identified distinct spatial domains wherein tumor cell proliferation occurs. Importantly, the vascular niche was closely associated with a population of CD44-expressing macrophages enriched for a proangiogenic gene signature. Taken together, this study provides insights into the spatial heterogeneity of PDAC and suggests a role for CD44-expressing macrophages in shaping the vascular niche. Significance: Imaging mass cytometry revealed that pancreatic ductal cancers are composed of 10 distinct cellular neighborhoods, including a vascular niche enriched for macrophages expressing high levels of CD44 and a proangiogenic gene signature.

Yeast-derived particulate beta-glucan induced angiogenesis via regulating PI3K/Src and ERK1/2 signaling pathway

The importance of β-glucan from S. cerevisiae in angiogenesis has not been well studied. We investigated whether β-glucan induces angiogenesis through PI3K/Src and ERK1/2 signaling pathway in HUVECs. We identified that β-glucan induced phosphorylation of PI3K, Src, Akt, eNOS, and ERK1/2. Subsequently, we found that this phosphorylation increased the viability of HUVECs. We also observed that stimulation of β-glucan promoted the activity of MEF2 and MEF2-dependent pro-angiogenic genes, including EGR2, EGR3, KLF2, and KLF4. Additionally, the role of β-glucan in angiogenesis was confirmed using in vitro and ex vivo experiments including cell migration, capillary-like tube formation and mouse aorta ring assays. To determine the effect of β-glucan on the PI3K/Akt/eNOS and ERK1/2 signaling pathway, PI3K inhibitor wortmannin and ERK1/2 inhibitor SCH772984 were used. Through the Matrigel plug assay, we confirmed that β-glucan significantly increased angiogenesis in vivo. Taken together, our study demonstrates that β-glucan promotes angiogenesis via through PI3K and ERK1/2 signaling pathway.

Abstract 5386: Increased sprouting angiogenesis in a microphysiological model of cisplatin-resistant recurrent lung cancers

Abstract Non-small cell lung cancer (NSCLC) constitutes 85% of total lung cancer cases and first-line therapies still often employ the platinum-based agents. Cisplatin therapy for NSCLC often leads to relapse of aggressive tumors with high metastatic potential after a period of dormancy. We hypothesize that cisplatin-resistant tumor cell phenotypes will increase features of aggressivity including sprouting angiogenesis in microphysiological models of NSCLC. First, we engineered various TME configurations using A549 cell spheroids and cancer associated fibroblasts (CAF) to establish a baseline of tumor cell proliferation, ECM deposition, and expression of proangiogenic and proinflammatory genes. Next, we generated cisplatin-resistant (Cis-R) A549 sub-lines to test the hypothesis that 3D tumor models engineered using these drug-resistant cells will capture hallmarks of highly aggressive recurrent tumors such as increased expression of progression-associated genes, increased induction of local stromal reactions and more profound angiogenesis. We treated parent A549 with 25 μM cisplatin for 96 hours followed by at least 14 days of recovery to generate Cis-R A549 cells. Interestingly, Cis-R spheroids exhibited significantly lower rates of proliferation (P<0.01) relative to parent A549 spheroids as measured by Ki67 index. Cis-R cells and spheroids exhibit increased genetic expression for genes involved in inflammation, EMT, proliferation and chemoresistance. Furthermore, Cis-R cells in 2D displayed a mesenchymal-like morphology and further exhibited dysmorphic spheroid morphology in 3D. We then developed a membrane free microphysiological system (MPS) comprised of two adjacent and contiguous tissue layers to investigate cancer mediated angiogenesis. We seeded one layer with lung fibroblasts and endothelial cells, which formed a vascularized network, while the other lane was cultured with no cells (control), parent A549 spheroids, or Cis-R A549 spheroids. Media void of VEGF was used throughout the duration of the experiment to isolate the effect of TME-derived factors. We measured increased angiogenetic sprouting induced by Cis-R tissues relative to parent using the following metrics: sprout density (Cis-R = 49.86 ± 10.93 µm/µm2, parent = 13.73 ± 4.12 µm/µm2, blank = 14.76 ± 4.22 µm/µm2), sprout length (Cis-R = 15006 ± 5507 µm, parent = 2466 ± 639.1 µm, blank = 4749 ± 1547 µm), and sprout area fraction (Cis-R = 0.10 ± 0.028, parent = 0.041 ± 0.010, blank = 0.032 ± 0.0095). Thus, the creation of drug resistant tumor cell sub lines enables the construction of more aggressive engineered tumor microenvironments. Future iterations of our MPS model will be used to investigate angiogenesis in the context of additional cancers, including breast and colon, while transitioning to the use of patient-derived organoids. Citation Format: Elisabet Olsen, G. Wills Kpeli, Omar M.K. Ahmad, Mark Mondrinos. Increased sprouting angiogenesis in a microphysiological model of cisplatin-resistant recurrent lung cancers [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 5386.

Cancer-associated Fibroblast-specific Expression of the Matricellular Protein CCN1 Coordinates Neovascularization and Stroma Deposition in Melanoma Metastasis.

In human patients, the expression of proangiogenic matricellular protein CCN1 in CAFs correlates positively with expression of stroma and angiogenic markers and progressive disease/resistance to checkpoint inhibitor therapy. In an animal model, loss of CCN1 from CAFs impaired metastasis of melanoma cells, neovascularization, and collagen deposition, emphasizing that CAFs coordinate cellular behavior in a tumor microenvironment and that CCN1 may be a novel target.

Hypoxic bone marrow mesenchymal stem cell exosomes promote angiogenesis and enhance endometrial injury repair through the miR-424-5p-mediated DLL4/Notch signaling pathway.

Currently, bone marrow mesenchymal stem cells (BMSCs) have been reported to promote endometrial regeneration in rat models of mechanically injury-induced uterine adhesions (IUAs), but the therapeutic effects and mechanisms of hypoxic BMSC-derived exosomes on IUAs have not been elucidated. To investigate the potential mechanism by which the BMSCS-derived exosomal miR-424-5p regulates IUA angiogenesis through the DLL4/Notch signaling pathway under hypoxic conditions and promotes endometrial injury repair. The morphology of the exosomes was observed via transmission electron microscopy, and the expression of exosome markers (CD9, CD63, CD81, and HSP70) was detected via flow cytometry and Western blotting. The expression of angiogenesis-related genes (Ang1, Flk1, Vash1, and TSP1) was detected via RT‒qPCR, and the expression of DLL4/Notch signaling pathway-related proteins (DLL4, Notch1, and Notch2) was detected via Western blotting. Cell proliferation was detected by a CCK-8 assay, and angiogenesis was assessed via an angiogenesis assay. The expression of CD3 was detected by immunofluorescence. The endometrial lesions of IUA rats were observed via HE staining, and the expression of CD3 and VEGFA was detected via immunohistochemistry. Compared with those in exosomes from normoxic conditions, miR-424-5p was more highly expressed in the exosomes from hypoxic BMSCs. Compared with those in normoxic BMSC-derived exosomes, the proliferation and angiogenesis of HUVECs were significantly enhanced after treatment with hypoxic BMSC-derived exosomes, and these effects were weakened after inhibition of miR-424-5p. miR-424-5p can target and negatively regulate the expression of DLL4, promote the expression of the proangiogenic genes Ang1 and Flk1, and inhibit the expression of the antiangiogenic genes Vash1 and TSP1. The effect of miR-424-5p can be reversed by overexpression of DLL4. In IUA rats, treatment with hypoxic BMSC exosomes and the miR-424-5p mimic promoted angiogenesis and improved endometrial damage. The hypoxic BMSC-derived exosomal miR-424-5p promoted angiogenesis and improved endometrial injury repair by regulating the DLL4/Notch signaling pathway, which provides a new idea for the treatment of IUAs.

Abstract TMP5: Mutation of Pdgfrβ Enhances the Severity of Brain Arteriovenous Malformation on Endoglin Mutant Mice Through Exacerbation of Angiogenesis and Inflammation

Background and Purpose: Brain arteriovenous malformations (bAVMs) are tangles of abnormal vessels shunting blood directly from arteries to veins. Reduction of pericytes is correlated with bAVM hemorrhage. The PDGFB/PDGFRβ signaling plays important roles in regulating pericyte recruitment during angiogenesis. Hypothesis: Mutation of PDGFRβ causes cerebrovascular malformation and enhances bAVM severity in endoglin ( Eng ) deficient mice. Methods: Three mouse models were used: (1) Pdgfrβ F7 (F7) mice that have mutations disrupting most Pdgfrβ signaling, (2) Pdgfb icreER; Eng f/f mice that have Pdgfb promoter driving, tamoxifen (TM) inducible cre expression in endothelial cells (ECs) and a floxed Eng gene; and (3) PdgfbicreER; Eng f/f ;F7 +/- mice were used. Brain angiogenesis was induced by intra-parenchymal injection of AAV-VEGF. BAVMs were induced in PdgfbicreER; Eng f/f and PdgfbicreER; Eng f/f ;F7 +/- mice by inducting EC Eng deletion through TM treatment and brain angiogenesis through intra-brain injection of AAV-VEGF. BAVM phenotypes were analyzed 8-weeks after model induction by latex vascular cast to detect arteriovenous shunts, immunostaining to analyze vessel morphology, Prussian blue staining to quantify microhemorrhage and RNAseq to investigate gene expression profiles. Results: Dysplastic vessels with reduced pericyte coverage were detected in the brain and brain angiogenic region of F7 +/- and F7 +/+ mice. AVM like vessels were detected in the brain of 16% F7 +/- and 66% of F7 +/+ mice. F7 mutation increases bAVM penetrance in Eng deficient mice (71% vs 50%), dysplastic vessels and microhemorrhage. RNAseq data revealed that mutation of Pdgfrb increased the expression of pro-angiogenic and endothelial cell proliferation genes and genes promote cell migration and inflammation in the bAVMs of Eng mutant mice. Conclusion: Pdgfrβ mutation exacerbates the severity of bAVM in Eng mutant mice by enhancing angiogenesis and inflammation.

Oral Metformin Inhibits Choroidal Neovascularization by Modulating the Gut-Retina Axis.

Emerging data indicate that metformin may prevent the development of age-related macular degeneration (AMD). Whereas the underlying mechanisms of metformin's anti-aging properties remain undetermined, one proposed avenue is the gut microbiome. Using the laser-induced choroidal neovascularization (CNV) model, we investigate the effects of oral metformin on CNV, retinal pigment epithelium (RPE)/choroid transcriptome, and gut microbiota. Specific pathogen free (SPF) male mice were treated via daily oral gavage of metformin 300mg/kg or vehicle. Male mice were selected to minimize sex-specific differences to laser induction and response to metformin. Laser-induced CNV size and macrophage/microglial infiltration were assessed by isolectin and Iba1 immunostaining. High-throughput RNA-seq of the RPE/choroid was performed using Illumina. Fecal pellets were analyzed for gut microbiota composition/pathways with 16S rRNA sequencing/shotgun metagenomics, as well as microbial-derived metabolites, including small-chain fatty acids and bile acids. Investigation was repeated in metformin-treated germ-free (GF) mice and antibiotic-treated/GF mice receiving fecal microbiota transplantation (FMT) from metformin-treated SPF mice. Metformin treatment reduced CNV size (P < 0.01) and decreased Iba1+ macrophage/microglial infiltration (P < 0.005). One hundred forty-five differentially expressed genes were identified in the metformin-treated group (P < 0.05) with a downregulation in pro-angiogenic genes Tie1, Pgf, and Gata2. Furthermore, metformin altered the gut microbiome in favor of Bifidobacterium and Akkermansia, with a significant increase in fecal levels of butyrate, succinate, and cholic acid. Metformin did not suppress CNV in GF mice but colonization of microbiome-depleted mice with metformin-derived FMT suppressed CNV. These data suggest that oral metformin suppresses CNV, the hallmark lesion of advanced neovascular AMD, via gut microbiome modulation.

IL-21 promoting angiogenesis contributes to the development of psoriasis.

Elevated IL-21 expression which can effectively induce Th17 cell differentiation has been implicated in the pathogenesis of psoriasis, but its role in angiogenesis remains poorly understood. PASI and PSI score assessment was applied to evaluate the severity of psoriatic lesions. The expression of IL-21, IL-21 receptor (IL-21R), CD31, VEGFA, MMP-9, and ICAM-1 in skin was determined by immunohistochemistry or quantitative real-time polymerase chain reaction. The serum level of IL-21 was measured by enzyme-linked immunosorbent assay (ELISA). Then, their correlation was analyzed statistically. Human umbilical vein endothelial cells (HUVECs) cocultured with conditional medium from normal human epidermal keratinocytes (NHEKs) were treated with IL-21 and/or M5 cocktail (mixture of IL-1α, IL-17A, IL-22, TNF-α, and oncostatin M). The migration and tube formation of HUVECs were detected, and the levels of VEGFA, MMP-9, and ICAM-1 in NHEKs were measured by Western blotting or ELISA. Increased IL-21 and IL-21R expression was observed in psoriatic sera or skin specimens, with IL-21R mainly locating in keratinocytes and IL-21 in immune cells. Pearson analysis showed significantly positive correlation between IL-21/IL-21R and erythema scores/microvessel density in psoriatic lesions. Moreover, the expression of proangiogenic genes, VEGFA, ICAM-1, and MMP-9 was upregulated in skins of psoriasis. Additionally, in M5 microenvironment, migration and tube formation could be magnified in HUVECs using IL-21 pre-treated NHEK medium. Mechanically, the co-stimulation of IL-21 and M5 to NEHKs increased the expression of ICAM-1. IL-21 could regulate keratinocytes to secrete ICAM-1, thereby promoting angiogenesis in psoriasis.

Single-cell transcriptomics identifies adipose tissue CD271+ progenitors for enhanced angiogenesis in limb ischemia

Therapeutic angiogenesis using mesenchymal stem/stromal cell grafts have shown modest and controversial effects in preventing amputation for patients with critical limb ischemia. Through single-cell transcriptomic analysis of human tissues, we identify CD271+ progenitors specifically from subcutaneous adipose tissue (AT) as having the most prominent pro-angiogenic gene profile distinct from other stem cell populations. AT-CD271+ progenitors demonstrate robust invivo angiogenic capacity over conventional adipose stromal cell grafts, characterized by long-term engraftment, augmented tissue regeneration, and significant recovery of blood flow in a xenograft model of limb ischemia. Mechanistically, the angiogenic capacity of CD271+ progenitors is dependent on functional CD271 and mTOR signaling. Notably, the number and angiogenic capacity of CD271+ progenitors are strikingly reduced in insulin-resistant donors. Our study highlights the identification of AT-CD271+ progenitors with invivo superior efficacy for limb ischemia. Furthermore, we showcase comprehensive single-cell transcriptomics strategies for identification of suitable grafts for cell therapy.