Angiogenic Cells Research Articles

ANGI-03. GLIOMA STEM CELLS INDUCE VASECTASIA – A NON-ANGIOGENIC BRAIN TUMOUR NEOVASCULARIZATION PROCESS DRIVEN BY INTERCELLULAR EGFR TRANSFER THROUGH EXTRACELLULAR VESICLES

Abstract Tumour neovascularization in glioblastoma (GBM) is abundant, abnormal and poorly understood, and it likely involves multiple co-existing mechnanisms with distinct modes of regulation. Since glioma stem cells (GSCs) engage in bidirectional interactions with the vasculature we dissected the resulting vascular responses elicited by either proneural (PN), or mesenchymal (MES) GSC subtypes established from patients isolates and able to drive progression of orthotopic intracranial xenografts in mice. We report here that while PN-GSCs elicited mostly angiogenesis-type vascular capillary growth processes, their MES-GSC counterparts elicited non-angiogenic growth of enlarged vessels (vasectesia), paradoxically associated with reduced blood vessel density. Vasectasia was dependent on the expression of oncogenic EGFR/EGFRvIII by MES-GSCs, and on the related kinase/signalling activity, as EGFR/EGFRvIII gene disruption and Dacomitinib treatment suppressed formation of large blood vessels in vivo and resulted in a preponderance of a small calliber (angiogenic) vasculature throughout xenografts. Notably, the analysis of spatial transcriptomes and single cell RNA sequencing revealed distinct molecular traits of endothelial cells asociated with vasectasia and angiogesis, the former enriched in Birc5, but depleted for angiogenic tip cell markers (Vegfr2, Apln). Mechnaistically, vasectasia was linked to the intercellular transfer of EGFR from MES-GSCs to endothelial cells resulting in ectopic activation of EGFR signalling in the endothelial cell bacground, both in vitro and in a model of in vivo blood vessel recruitment into implanted matrigel pellets. Simultaneous pharmacological blockade of both, angiogenesis (anti-VEGR2) and vasectasia (Dacomitinib) lead to a marked increase in survival of mice with aggressive GBM lesions initiated by MES-GSCs. We suggest that, vasectasia driven by intercellular transfer of oncogenic EGFR may represent a new, subtype-specific mechanism of GBM neovascularization, and a plausible target for agents aiming to modify vascular tumour microenvironment.

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

Abstract Introduction Augmenting evidence indicates that long noncoding RNAs (lncRNAs) are playing a crucial role in diverse cellular/pathological processes. Intercellular transfer of extracellular vesicles (EVs) transmitted lncRNA regulates vascular health and diseases. However, whether lncRNA expression in EVs is regulated in patients with coronary artery disease, is unknown. A PCR-based lncRNA array analysis revealed that EV-PUNISHER was significantly upregulated in patients with CAD (n=221) compared to healthy subjects. Our data showed that PUNISHER is the most upregulated lncRNA in CAD in accordance with other highly expressed lncRNAs (FGF14-AS2, HYMAI, Gas5, Malat1, etc.). SiRNA-mediated silencing in ECs revealed that depletion of PUNISHER suppresses the migration and proliferation of ECs. Depletion of PUNISHER decreases cell survival by reducing cell viability and proliferation through increased apoptosis and cytotoxicity. To investigate EC function in PUNISHER depleted cells, sprouting and tube formation assay revealed that PUNISHER is an important mediator EC functions, which acts as an anti-angiogenic factor. In vitro atherosclerotic stimuli (OxLDL, TNF-α, IL-6) increased PUNISHER expression in EV/EC in a dose-dependent way. Microarray analysis identified a series of pro- and anti-angiogenic genes as well genes directly involved in cell viability that are differentially regulated. By using lncRNA-FISH and vesicle degradation assays, we showed that PUNISHER is incorporated into EMVs, augmented recipient EC function in vitro upon transfer via EMVs. To examine whether EMV-PUNISHER promotes the EC function, different in vitro functional experiments with ECs (tube formation, angiogenic sprouting, migration, proliferation, apoptosis, etc.) confirmed that PUNISHER is an important regulator. MS analysis has revealed EMV contains numerous proteins, including RNA binding proteins such as hnRNPU, hnRNPK, hnRNPA2/B1, etc. By using RNA-pulldown and RNA immunoprecipitation, we confirmed that PUNISHER interacts with hnRNPU, which facilitates packaging into EMV prior to transfer to recipient EC. The interaction acts as an important positive regulator of cell viability and survival of recipient cells, identified using functional assay (migration, viability, proliferation, and angiogenesis). Transcription factor array has shown that PUNISHER regulates NFkB to control cellular viability and apoptosis. A murine re-endothelialization model after injection of 1×107 EVs or bulk ncRNAs (1, 5, 10 nM) revealed that EV-PUNISHER promotes reendothelialization. Ex vivo internalization/uptake of PKH-67-labeled EVs revealed that EVs are detectable in the cross-section of EV-perfused carotid artery. Our study unveiled for the first time that EV-incorporated PUNISHER exerts its function in EC which might be beneficial in cardiovascular pathologies. Our data indicate that PUNISHER can be targeted to develop targeted therapeutics.

Effects of mesenchymal stromal cells and human recombinant Nerve Growth Factor delivered by bioengineered human corneal lenticule on an innovative model of diabetic retinopathy.

Diabetic retinopathy (DR) is a microvascular complication of diabetes in which neurodegeneration has been recently identified as a driving force. In the last years, mesenchymal stromal cells (MSCs) and neurotrophins like Nerve Growth Factor (NGF), have garnered significant attention as innovative therapeutic approaches targeting DR-associated neurodegeneration. However, delivering neurotrophic factors directly in the eye remains a challenge. Hence, this study evaluated the effects of MSCs from human amniotic fluids (hAFSCs) and recombinant human NGF (rhNGF) delivered by human corneal lenticule (hCL) on a high glucose (HG) induced ex vivo model simulating the molecular mechanisms driving DR. Porcine neuroretinal explants exposed to HG (25 mM for four days) were used to mimic DR ex vivo. hCLs collected from donors undergoing refractive surgery were decellularized using 0.1% sodium dodecyl sulfate and then bioengineered with hAFSCs, microparticles loaded with rhNGF (rhNGF-PLGA-MPs), or both simultaneously. Immunofluorescence (IF) and scanning electron microscopy (SEM) analyses were performed to confirm the hCLs bioengineering process. To assess the effects of hAFSCs and rhNGF, bioengineered hCLs were co-cultured with HG-treated neuroretinal explants and following four days RT-PCR and cytokine array experiments for inflammatory, oxidative, apoptotic, angiogenic and retinal cells markers were performed. Data revealed that HG-treated neuroretinal explants exhibit a characteristic DR-phenotype, including increased level of NF-kB, NOS2, NRF2 GFAP, VEGFA, Bax/Bcl2 ratio and decreased expression of TUBB3 and Rho. Then, the feasibility to bioengineer decellularized hCLs with hAFSCs and rhNGF was demonstrated. Interestingly, co-culturing hAFSCs- and rhNGF- bioengineered hCLs with HG-treated neuroretinal explants for four days significantly reduced the expression of inflammatory, oxidative, apoptotic, angiogenic and increased retinal markers. Overall, we found for the first time that hAFSCs and rhNGF were able to modulate the molecular mechanisms involved in DR and that bioengineered hCLs represents a promising ocular drug delivery system of hAFSCs and rhNGF for eye diseases treatment. In addition, results demonstrated that porcine neuroretinal explants treated with HG is a useful model to reproduce ex vivo the DR pathophysiology.

Circulating CD31+ Angiogenic T cells are reduced in prediabetes and increase with exercise training

AimsTo investigate circulating angiogenic cells in adults with prediabetes and the effect of a structured exercise program. MethodsA cohort of adults with overweight/obesity and either normal glucose (NG) or prediabetes were randomised to receive exercise (Exercise) (as twice weekly supervised combined high intensity aerobic exercise and progressive resistance training, and once weekly home-based aerobic exercise) or an unsupervised stretching intervention (Control) for 12 weeks. Circulating angiogenic T cells, muscle strength, and cardiovascular disease risk factors, including blood lipids, arterial stiffness, central haemodynamic responses, and cardiorespiratory fitness (VO2peak) in those with prediabetes (n = 35, 16 Control, 19 Exercise) and NG (n = 37, 17 Control, 20 Exercise) were analysed at baseline and after the 12-week intervention. ResultsAt baseline, compared with NG those with prediabetes demonstrated reduced VO2peak, angiogenic CD31+CD8+ T cells and VEGFR2+CD4+ T cells, and increased systolic blood pressure. CD31+ T cells were negatively correlated with cardiovascular disease (CVD) risk. Compared with Control, exercise training increased muscle strength, VO2peak, and CD31+CD4+ and CD31+CD8+ T cells in NG and prediabetes. ConclusionsCirculating angiogenic CD31+ T cells are decreased in people with prediabetes and are enhanced with exercise training. Exercise increases CD31+ T cells, and through this mechanism it is proposed that it may reduce CVD risk. Trial registrationAustralian New Zealand Clinical Trials Registry number: ACTRN12617000552381.

Abstract P205: Single-nucleus Sequencing Reveals Endothelial Cell-specific Dysregulation in the Kidney in Multiple Models of Hypertension

The kidney plays a crucial role in the onset and progression of hypertension. To investigate the roles of different renal cell types in hypertension, we applied single-nucleus sequencing and spatial transcriptome sequencing to kidney samples from three well-established hypertension models: C57BL/6 mice treated with Ang II, the Dahl salt-sensitive (SS) rat on high-salt diets, and the spontaneously hypertensive rat (SHR), along with their respective controls including Sprague-Dawley (SD) and Wistar Kyoto (WKY) rats. Our dataset comprised 179,637 nuclei from 24 samples across 12 experimental conditions, identifying 17 major cell types including various epithelial cells, endothelial cells, vascular smooth muscle cells, fibroblasts, and immune cells. At baseline, differentially expressed genes between the SS and SD groups were specifically enriched in endothelial cell pathways related to vascular development. This included expression deficiencies in genes involved in angiogenesis in SS rats, such as Arhgap24 and Nox4. Compensatory expression changes in these genes were observed in the SS rats after a high-salt diet and in 26-week-old SHR, but not in their respective controls. These genes were also associated with multiple blood pressure-associated single-nucleotide polymorphisms (SNPs). Furthermore, changes in endothelial cell communication were most prominent upon stimuli, with impaired PTPRM (Protein Tyrosine Phosphatase Receptor Type M) signaling being a robust feature in hypertension models, involving angiogenic endothelial cells in the SS model and proliferating capillary endothelial cells in the SHR model. Our study highlights the critical role of renal endothelial cell angiogenic dysfunction in the pathogenesis of hypertension and suggests a link between specific renal endothelial cell subtype dysregulation and hypertension development.

Cerebral hypoperfusion exacerbates vascular dysfunction after traumatic brain injury

Traumatic brain injuries are extremely common, and although most patients recover from their injuries many TBI patients suffer prolonged symptoms and remain at a higher risk for developing cardiovascular disease and neurodegeneration. Moreover, it remains challenging to identify predictors of poor long-term outcomes. Here, we tested the hypothesis that preexisting cerebrovascular impairment exacerbates metabolic and vascular dysfunction and leads to worse outcomes after TBI. Male mice underwent a mild surgical reduction in cerebral blood flow using a model of bilateral carotid artery stenosis (BCAS) wherein steel microcoils were implanted around the carotid arteries. Then, 30 days post coil implantation, mice underwent TBI or sham surgery. Gene expression profiles, cerebral blood flow, metabolic function, oxidative damage, vascular health and angiogenesis were assessed. Single nuclei RNA sequencing of endothelial cells isolated from mice after TBI showed differential gene expression profiles after TBI and BCAS, that were further altered when mice underwent both challenges. TBI but not BCAS increased mitochondrial oxidative metabolism. Both BCAS and TBI decreased cerebrovascular responses to repeated whisker stimulation. BCAS induced oxidative damage and inflammation in the vasculature as well as loss of vascular density, and reduced the numbers of angiogenic tip cells. Finally, intravascular protein accumulation was increased among mice that experienced both BCAS and TBI. Overall, our findings reveal that a prior vascular impairment significantly alters the profile of vascular health and function of the cerebrovasculature, and when combined with TBI may result in worsened outcomes.

Molecular Changes Implicate Angiogenesis and Arterial Remodeling in Systemic Sclerosis-Associated and Idiopathic Pulmonary Hypertension.

Pulmonary hypertension (PH) is a common complication of systemic sclerosis (SSc) and a leading cause of mortality among patients with this disease. PH can also occur as an idiopathic condition (idiopathic pulmonary arterial hypertension). Investigation of transcriptomic alterations in vascular populations is critical to elucidating cellular mechanisms underlying pathobiology of SSc-associated and idiopathic PH. We analyzed single-cell RNA sequencing profiles of endothelial and perivascular mesenchymal populations from explanted lung tissue of patients with SSc-associated PH (n=16), idiopathic pulmonary arterial hypertension (n=3), and healthy controls (n=15). Findings were validated by immunofluorescence staining of explanted human lung tissue. Three disease-associated endothelial populations emerged. Two angiogenic endothelial cell (EC) subtypes markedly expanded in SSc-associated PH lungs: tip ECs expressing canonical tip markers PGF and APLN and phalanx ECs expressing genes associated with vascular development, endothelial barrier integrity, and Notch signaling. Gene regulatory network analysis suggested enrichment of Smad1 (SMAD family member 1) and PPAR-γ (peroxisome proliferator-activated receptor-γ) regulon activities in these 2 populations, respectively. Mapping of potential ligand-receptor interactions highlighted Notch, apelin-APJ (apelin receptor), and angiopoietin-Tie (tyrosine kinase with immunoglobulin-like and EGF-like domains 1) signaling pathways between angiogenic ECs and perivascular cells. Transitional cells, expressing both endothelial and pericyte/smooth muscle cell markers, provided evidence for the presence of endothelial-to-mesenchymal transition. Transcriptional programs associated with arterial endothelial dysfunction implicated VEGF-A (vascular endothelial growth factor-A), TGF-β1 (transforming growth factor beta-1), angiotensin, and TNFSF12 (tumor necrosis factor ligand superfamily member 12)/TWEAK (TNF-related weak inducer of apoptosis) in the injury/remodeling phenotype of PH arterial ECs. These data provide high-resolution insights into the complexity and plasticity of the pulmonary endothelium in SSc-associated PH and idiopathic pulmonary arterial hypertension and provide direct molecular insights into soluble mediators and transcription factors driving PH vasculopathy.

Injectable hydrogel scaffold incorporating microspheres containing cobalt-doped bioactive glass for bone healing.

Injectable in situ-forming scaffolds that induce both angiogenesis and osteogenesis have been proven to be promising for bone healing applications. Here, we report the synthesis of an injectable hydrogel containing cobalt-doped bioactive glass (BG)-loaded microspheres. Silk fibroin (SF)/gelatin microspheres containing BG particles were fabricated through microfluidics. The microspheres were mixed in an injectable alginate solution, which formed an in situ hydrogel by adding CaCl2. The hydrogel was evaluated for its physicochemical properties, in vitro interactions with osteoblast-like and endothelial cells, and bone healing potential in a rat model of calvarial defect. The microspheres were well-dispersed in the hydrogel and formed pores of >100 μm. The hydrogel displayed shear-thinning behavior and modulated the cobalt release so that the optimal cobalt concentration for angiogenic stimulation, cell proliferation, and deposition of mineralized matrix was only achieved by the scaffold that contained BG doped with 5% wt/wt cobalt (A-S-G5Co). In the scaffold containing higher cobalt content, a reduced biomimetic mineralization on the surface was observed. The gene expression study indicated an upregulation of the osteogenic genes of COL1A1, ALPL, OCN, and RUNX2 and angiogenic genes of HIF1A and VEGF at different time points in the cells cultured with the A-S-G5Co. Finally, the in vivo study demonstrated that A-S-G5Co significantly promoted both angiogenesis and osteogenesis and improved bone healing after 12 weeks of follow-up. These results show that incorporation of SF/gelatin microspheres containing cobalt-doped BG in an injectable in situ-forming scaffold can effectively enhance its bone healing potential through promotion of angiogenesis and osteogenesis.

Modulating Tumor Immunity by Targeting Tumor Fibrotic Stroma and Angiogenic Vessels for Lung Cancer Treatment.

Fibrotic stroma and angiogenic tumor vessels play an important role in modulating tumor immunity. We previously reported a rationally designed protein (ProAgio) that targets integrin αvβ3 at a novel site. ProAgio induces the apoptosis of cells that express high levels of the integrin. Both activated cancer-associated fibroblasts (CAFs) and angiogenic endothelial cells (aECs) in tumors express high levels of integrin αvβ3. ProAgio simultaneously and specifically induces apoptosis in CAFs and aECs in tumors. We provide evidence here that the depletion of CAFs and the elimination of leaky tumor angiogenic vessels by ProAgio alter tumor immunity. ProAgio reduces CD4+ Treg and Myeloid-derived suppressor cells (MDSCs), increases CD8+ T-cells, and increases the M1/M2 macrophage ratio in the tumor. The depletion of dense fibrotic stroma (CAFs) by ProAgio decreases the Programmed Death Ligand 1 (PDL-1) levels in the stroma areas surrounding the tumors, and thus strongly increases the delivery of anti-PDL-1 antibody to the target cancer cells. The impact of ProAgio on tumor immunity provides strong synergistical effects of checkpoint inhibitors on lung cancer treatment.

P-801 Sub-endometrial administration of angiogenic precursor cells and growth factors effectively optimizes endometrial thickness (EMT) and pregnancy outcomes in women with refractory thin endometrium

Abstract Study question Can hysteroscopic instillation of autologous blood derived progenitor cells combined with platelet derived growth factor concentrate into subendometrial region help patient with thin endometrium conceive? Summary answer Hysteroscopic subendometrial administration of stem cells represents a safe effective alternative method for patients recommended surrogacy, fail to improve despite using all possible treatment options What is known already Chronically thin endometrium is a challenge in ART, results in repeated expensive IVF cycles, cycle cancellations, unplanned cryopreservation of embryos and, surrogacy. Many studies have successfully used stem cells or platelet derivatives to rejuvenate reproductive tissues. When these growth-promoting cells and growth factors from platelets are delivered into basal layer which is origin of endometrial cells quality of the endometrium can be improved. While bone marrow is excellent source for obtaining stem cells main challenge is invasiveness of collection. This forms the basis for collecting mobilized endothelial progenitor cells from bone marrow into circulation for easy collection Study design, size, duration This study is to evaluate effect angiogenic precursor cells and growth factors derived from peripheral blood and bone marrow in improving endometrial quality and pregnancy outcomes. In the present pilot study involving 87 patients with persistent refractory thin endometrium were included and underwent frozen embryo transfer from April 2021-January 2024 at A4 fertility center, Chennai. All patients were treated with peripheral blood derived progenitor cells and platelet derived growth factors. Participants/materials, setting, methods 87 patients with EMT<7mm and >2 cancelled/failed cycles included. Two doses of SC-G-CSF were given on Menstrual Cycle Day 3&4, followed by venous blood aspiration on MCD-5. Seragen’s selective enrichment protocol was used to prepare circulating endothelial progenitor cells and growth factors concentrate. With 2.9mm hysteroscope and single lumen ovum pickup needle harvested cells and growth factors were injected on all four walls of cavity. Main results and the role of chance In this cohort, all patients presented refractory thin endometrium during hormone replacement therapy (HRT), despite exhaustive prior medication attempts. Subendometrial injection was employed, resulting in a significant increase in endometrial thickness (EMT) post peripheral blood cell injection (6.0 ± 0.71 vs. 7.80 ± 0.8; P = 0.0001), averaging a substantial improvement of 1.80mm. An optimal response, defined as EMT≥ 7mm, was observed in 47.2% (53) of cases. Patients with improved thickness underwent embryo transfer in the subsequent cycle, displaying a significant enhancement in endometrial thickness. Out of 53 patients, 47.2% (25) tested positive for β-HCG, while 45.3% (24) tested negative. Miscarriage occurred in 9.4% (5) of patients, with 8.8% (3) delivering healthy full-term babies. Notably, 28.3% (15) sustained uneventful pregnancies, accounting for a 34% sustained implantation rate. Comparatively, when benchmarked against subendometrial platelet-rich plasma (PRP) studies, primary and secondary outcomes in our study demonstrated superiority. This favorable outcome is attributed to the combined utilization of mobilized progenitor cells and growth factors. Prior research has established that the endometrium derives its stem cell content from the bone marrow, underscoring the efficacy of this novel approach. Limitations, reasons for caution Future research should explore avenues for women with refractory thin endometrium, offering conception options without resorting to surrogacy. Clinician expertise in patientselection, personalized dosage, and administration is crucial. While our study offers promising insights, its limitations include small sample size and lack of randomization, emphasizing need for larger, controlled trials. Wider implications of the findings Our study reveals that combine use of growth factors progenitor cells enhances endometrial thickness and improves pregnancy and live birth rates. Re-evaluating need for surrogacy in nonresponsive thin endometrium may be warranted. Insights from hematopoietic stem cell research offer reliable strategy for routine use in IVF clinical practice. Trial registration number Not applicable

MTORC1 Signaling in Brain Endothelial Progenitors Contributes to CCM Pathogenesis.

Cerebral vascular malformations (CCMs) are primarily found within the brain, where they result in increased risk for stroke, seizures, and focal neurological deficits. The unique feature of the brain vasculature is the blood-brain barrier formed by the brain neurovascular unit. Recent studies suggest that loss of CCM genes causes disruptions of blood-brain barrier integrity as the inciting events for CCM development. CCM lesions are proposed to be initially derived from a single clonal expansion of a subset of angiogenic venous capillary endothelial cells (ECs) and respective resident endothelial progenitor cells (EPCs). However, the critical signaling events in the subclass of brain ECs/EPCs for CCM lesion initiation and progression are unclear. Brain EC-specific CCM3-deficient (Pdcd10BECKO) mice were generated by crossing Pdcd10fl/fl mice with Mfsd2a-CreERT2 mice. Single-cell RNA-sequencing analyses were performed by the chromium single-cell platform (10× genomics). Cell clusters were annotated into EC subtypes based on visual inspection and GO analyses. Cerebral vessels were visualized by 2-photon in vivo imaging and tissue immunofluorescence analyses. Regulation of mTOR (mechanistic target of rapamycin) signaling by CCM3 and Cav1 (caveolin-1) was performed by cell biology and biochemical approaches. Single-cell RNA-sequencing analyses from P10 Pdcd10BECKO mice harboring visible CCM lesions identified upregulated CCM lesion signature and mitotic EC clusters but decreased blood-brain barrier-associated EC clusters. However, a unique EPC cluster with high expression levels of stem cell markers enriched with mTOR signaling was identified from early stages of the P6 Pdcd10BECKO brain. Indeed, mTOR signaling was upregulated in both mouse and human CCM lesions. Genetic deficiency of Raptor (regulatory-associated protein of mTOR), but not of Rictor (rapamycin-insensitive companion of mTOR), prevented CCM lesion formation in the Pdcd10BECKO model. Importantly, the mTORC1 (mTOR complex 1) pharmacological inhibitor rapamycin suppressed EPC proliferation and ameliorated CCM pathogenesis in Pdcd10BECKO mice. Mechanistic studies suggested that Cav1/caveolae increased in CCM3-depleted EPC-mediated intracellular trafficking and complex formation of the mTORC1 signaling proteins. CCM3 is critical for maintaining blood-brain barrier integrity and CCM3 loss-induced mTORC1 signaling in brain EPCs initiates and facilitates CCM pathogenesis.

Fabricating Multiphasic Angiogenic Scaffolds Using Amyloid/Roxadustat-Assisted High-Temperature Protein Printing.

Repairing multiphasic defects is cumbersome. This study presents new soft and hard scaffold designs aimed at facilitating the regeneration of multiphasic defects by enhancing angiogenesis and improving cell attachment. Here, the nonimmunogenic, nontoxic, and cost-effective human serum albumin (HSA) fibril (HSA-F) was used to fabricate thermostable (up to 90 °C) and hard printable polymers. Additionally, using a 10.0 mg/mL HSA-F, an innovative hydrogel was synthesized in a mixture with 2.0% chitosan-conjugated arginine, which can gel in a cell-friendly and pH physiological environment (pH 7.4). The presence of HSA-F in both hard and soft scaffolds led to an increase in significant attachment of the scaffolds to the human periodontal ligament fibroblast (PDLF), human umbilical vein endothelial cell (HUVEC), and human osteoblast. Further studies showed that migration (up to 157%), proliferation (up to 400%), and metabolism (up to 210%) of these cells have also improved in the direction of tissue repair. By examining different in vitro and ex ovo experiments, we observed that the final multiphasic scaffold can increase blood vessel density in the process of per-vascularization as well as angiogenesis. By providing a coculture environment including PDLF and HUVEC, important cross-talk between these two cells prevails in the presence of roxadustat drug, a proangiogenic in this study. In vitro and ex ovo results demonstrated significant enhancements in the angiogenic response and cell attachment, indicating the effectiveness of the proposed design. This approach holds promise for the regeneration of complex tissue defects by providing a conducive environment for vascularization and cellular integration, thus promoting tissue healing.

Mammary adipocytes promote breast tumor cell invasion and angiogenesis in the context of menopause and obesity

The mechanism(s) underlying obesity-related postmenopausal (PM) breast cancer (BC) are not clearly understood. We hypothesized that the increased local presence of ‘obese’ mammary adipocytes within the BC microenvironment promotes the acquisition of an invasive and angiogenic BC cell phenotype and accelerates tumor proliferation and progression. BC cells, treated with primary mammary adipocyte secretome from premenopausal (Pre-M) and PM obese women (ObAdCM; obese adipocyte conditioned-media) upregulated the expression of several pro-tumorigenic factors including VEGF, lipocalin-2 and IL-6. Both Pre-M and PM ObAdCM stimulated endothelial cell recruitment and proliferation and significantly stimulated BC cell proliferation, migration and invasion. IL-6 and LCN2 induced STAT3/Akt signaling in BC cells and STAT3 inhibition abrogated the ObAdCM-stimulated BC cell proliferation and migration. Expression of proangiogenic regulators including VEGF, NRP1, NRP2, IL8RB, TGFβ2, and TSP-1 were found to be differentially regulated in mammary adipocytes from obese PM women. Comparative RNAseq indicated an upregulation of PI3K/Akt signaling, ECM-receptor interactions and lipid/fatty acid metabolism in PM versus Pre-M mammary adipocytes. Our results demonstrate that irrespective of menopausal status, cross-talk between obese mammary adipocytes and BC cells promotes tumor aggressiveness and suggest that targeting the LCN2/IL-6/STAT3 signaling axis may be a useful strategy in obesity-driven breast tumorigenesis.

Characterization of FOLH1 Expression in Renal Cell Carcinoma.

Given the emergence of PSMA-targeted diagnostic agents and therapeutics, we sought to investigate patterns of FOLH1 expression in RCC and their impacts on RCC outcomes. We conducted a pooled multi-institutional analysis of patients with RCC having undergone DNA and RNA next-generation sequencing. FOLH1-high/low expression was defined as the ≥75th/<25th percentile of RNA transcripts per million (TPM). Angiogenic, T-effector, and myeloid expression signatures were calculated using previously defined gene sets. Kaplan-Meier estimates were calculated from the time of tissue collection or therapy start. We included 1,724 patients in the analysis. FOLH1 expression was significantly higher in clear cell (71%) compared to non-clear cell RCC tumors (19.0 versus 3.3 TPM, p < 0.001) and varied by specimen site (45% primary kidney/55% metastasis, 13.6 versus 9.9 TPM, p < 0.001). FOLH1 expression was correlated with angiogenic gene expression (Spearman = 0.76, p < 0.001) and endothelial cell abundance (Spearman = 0.76, p < 0.001). While OS was similar in patients with FOLH1-high versus -low ccRCC, patients with FOLH1-high clear cell tumors experienced a longer time on cabozantinib treatment (9.7 versus 4.6 months, respectively, HR 0.57, 95% CI 0.35-0.93, p < 0.05). We observed differential patterns of FOLH1 expression based on histology and tumor site in RCC. FOLH1 was correlated with angiogenic gene expression, increased OS, and a longer duration of cabozantinib treatment.

The impact of acute and chronic aerobic and resistance exercise on stem cell mobilization: A review of effects in healthy and diseased individuals across different age groups

Stem cells (SCs) play a crucial role in tissue repair, regeneration, and maintaining physiological homeostasis. Exercise mobilizes and enhances the function of SCs. This review examines the effects of acute and chronic aerobic and resistance exercise on the population of SCs in healthy and diseased individuals across different age groups. Both acute intense exercise and moderate regular training increase circulating precursor cells CD34+ and, in particular, the subset of angiogenic progenitor cells (APCs) CD34+/KDR+. Conversely, chronic exercise training has conflicting effects on circulating CD34+ cells and their function, which are likely influenced by exercise dosage, the health status of the participants, and the methodologies employed. While acute activity promotes transient mobilization, regular exercise often leads to an increased number of progenitors and more sustainable functionality. Short interventions lasting 10–21 days mobilize CD34+/KDR + APCs in sedentary elderly individuals, indicating the inherent capacity of the body to rapidly activate tissue-reparative SCs during activity. However, further investigation is needed to determine the optimal exercise regimens for enhancing SC mobilization, elucidating the underlying mechanisms, and establishing functional benefits for health and disease prevention. Current evidence supports the integration of intense exercise with chronic training in exercise protocols aimed at activating the inherent regenerative potential through SC mobilization. The physical activity promotes endogenous repair processes, and research on exercise protocols that effectively mobilize SCs can provide innovative guidelines designed for lifelong tissue regeneration. An artificial neural network (ANN) was developed to estimate the effects of modifying elderly individuals and implementing chronic resistance exercise on stem cell mobilization and its impact on individuals and exercise. The network's predictions were validated using linear regression and found to be acceptable compared to experimental results.

Parenchymal cues define Vegfa-driven venous angiogenesis by activating a sprouting competent venous endothelial subtype

Formation of organo-typical vascular networks requires cross-talk between differentiating parenchymal cells and developing blood vessels. Here we identify a Vegfa driven venous sprouting process involving parenchymal to vein cross-talk regulating venous endothelial Vegfa signaling strength and subsequent formation of a specialized angiogenic cell, prefabricated with an intact lumen and pericyte coverage, termed L-Tip cell. L-Tip cell selection in the venous domain requires genetic interaction between vascular Aplnra and Kdrl in a subset of venous endothelial cells and exposure to parenchymal derived Vegfa and Apelin. Parenchymal Esm1 controls the spatial positioning of venous sprouting by fine-tuning local Vegfa availability. These findings may provide a conceptual framework for understanding how Vegfa generates organo-typical vascular networks based on the selection of competent endothelial cells, induced via spatio-temporal control of endothelial Kdrl signaling strength involving multiple parenchymal derived cues generated in a tissue dependent metabolic context.

A brain-specific angiogenic mechanism enabled by tip cell specialization

Vertebrate organs require locally adapted blood vessels1,2. The gain of such organotypic vessel specializations is often deemed to be molecularly unrelated to the process of organ vascularization. Here, opposing this model, we reveal a molecular mechanism for brain-specific angiogenesis that operates under the control of Wnt7a/b ligands—well-known blood–brain barrier maturation signals3–5. The control mechanism relies on Wnt7a/b-dependent expression of Mmp25, which we find is enriched in brain endothelial cells. CRISPR–Cas9 mutagenesis in zebrafish reveals that this poorly characterized glycosylphosphatidylinositol-anchored matrix metalloproteinase is selectively required in endothelial tip cells to enable their initial migration across the pial basement membrane lining the brain surface. Mechanistically, Mmp25 confers brain invasive competence by cleaving meningeal fibroblast-derived collagen IV α5/6 chains within a short non-collagenous region of the central helical part of the heterotrimer. After genetic interference with the pial basement membrane composition, the Wnt–β-catenin-dependent organotypic control of brain angiogenesis is lost, resulting in properly patterned, yet blood–brain-barrier-defective cerebrovasculatures. We reveal an organ-specific angiogenesis mechanism, shed light on tip cell mechanistic angiodiversity and thereby illustrate how organs, by imposing local constraints on angiogenic tip cells, can select vessels matching their distinctive physiological requirements.

Abstract 5384: The hypoxia-alleviating agent ITPP restores vascular function in zebrafish xenografts of pancreatic cancer

Abstract Pancreatic cancer mostly manifests as pancreatic ductal adenocarcinoma (PDAC) and is characterized by a highly hypoxic microenvironment that is associated with worse patient survival and resistance to therapy. Hypoxia induces angiogenesis and the resulting leaky and unstructured vessels fail to reoxygenate the tumor mass, thus exacerbating hypoxia and its downstream impact on tumorigenesis. Myo-inositol trispyrophosphate (ITPP) is a non-toxic allosteric effector of hemoglobin shown to reduce tumor growth and enhance survival in mice and rat models of different tumor types. Herein the aim is to assess for the first time the ability of ITPP to effectively treat PDAC tumors in vivo in zebrafish xenografts and to investigate its impact on vascular normalization. To that aim, four PDAC cell lines, AsPC1, Capan1, MIA PaCa2, and PANC1 were included, as well as the angiogenic breast cancer cell line, Hs578t. Cells were grown in appropriate conditions and on injection day, labeling was done with deep red. Labeled cells were then injected in the perivitelline space of anesthetized larvae. The following transgenic larvae were used: those having labeled blood vessels only (Tg(kdrl:DsRed)), labeled blood and lymphatic vessels (Tg(fli1:eGFP)), and double transgenics with labeled gata+ erythrocytes (Tg(fli1:eGFP;gata:dsRed)). One day post injection (dpi) xenografts were sorted based on size and treated by immersion for three consecutive days with control medium or ITPP at a concentration selected based on the maximum tolerated dose assay. Treatment was refreshed daily, and at 4dpi live confocal imaging was conducted on select Tg(fli1:eGFP;gata:dsRed) xenografts. All xenografts were then fixed and subjected to whole mount immunofluorescence. Nuclei were stained with DAPI and anti-Caspase3Asp175 was used as a marker for apoptosis. Imaging was performed using the Zeiss LSM confocal microscope and tumor size, apoptosis and mitotic figures were quantified with ImageJ. Vascular function was defined as the presence of gata+ cells within tumor-related vasculature, while vessel density and infiltration were determined in ImageJ by calculating the percentage of GFP area in the tumor. Thus far results from AsPC1-derived xenografts demonstrate that ITPP leads to a significant decrease in mitotic figures (p=0.0132) and an increase in apoptosis (p=0.0025), with no change in tumor size. Preliminary data reveal a trend of increased functional vasculature in both the AsPC1- and Hs578t- derived xenografts upon ITPP treatment. A trend of decreased vasculature infiltration could also be observed in the ITPP-treated Hs578t-derived xenografts. Based on these results, the zebrafish model could be an appropriate alternative for assessing the anti-tumorigenic activity of ITPP in PDAC in a short turnaround time and could potentially enable the visualization of ITPP’s impact on angiogenesis at single cell resolution. Citation Format: Raefa Abou Khouzam, Filipa Amorim, Ayesha Rifath, Husam Nawafleh, Vanda Povoa, Rania Faouzi Zaarour, Jean Marie Lehn, Rita Fior, Perparim Limani, Salem Chouaib. The hypoxia-alleviating agent ITPP restores vascular function in zebrafish xenografts of pancreatic cancer [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 5384.

Abstract 6851: The genomic, transcriptomic, and immunologic landscape of TEM8 (ANTXR1) in neuroendocrine neoplasms (NENs)

Abstract Introduction: TEM8 (coded by ANTXR1) is overexpressed in malignant tissues, with oncolytic viruses such as SVV-01 uniquely binding to this receptor on tumor-associated angiogenic endothelial cells, pericytes, fibroblasts, and immune inflammatory cells. Recent pre-clinical data suggest that TEM8-targeting therapies may convert immunologically “cold” tumor microenvironments (TME) into “hot” milieu more amenable to treatment with immune checkpoint inhibitors (ICIs). Methods: NextGen sequencing of DNA (592 genes or WES) and RNA (WTS) was performed on neuroendocrine neoplasms (NENs; N = 1724), excluding small-cell lung cancer, submitted to Caris Life Sciences (Phoenix, AZ). Mutations were defined as pathogenic SNVs/indels. ANTXR1 expression was divided by ANTXR1-expression quartiles (transcripts per million [TPM];Q4:H;Q1:L). PD-L1 expression (SP142; Positive (+): ≥2+,≥%5) was assessed by IHC. High tumor mutational burden (TMB-H) was defined as ≥10 mutations per MB. Cell infiltration in the TME was estimated by QuantiSEQ. Gene expression profiles were analyzed for transcriptional signatures predictive of response to immunotherapy (T cell-inflamed) and MAPK pathway activation score (MPAS). Overall survival (OS) data was obtained from insurance claims, and Kaplan-Meier estimates were calculated for molecularly defined subpopulations of patients. Mann-Whitney U and X2/Fisher-Exact tests were applied where appropriate, with p-values adjusted for multiple comparisons (p&amp;lt;0.05). Results: NENs from the adrenal gland had the highest median ANTXR1 expression (50 TPM), followed by those from the colon and rectum (43.5), genitourinary organs (43.3), and small bowel (excluding appendix) (39.2). The lowest median ANTXR1 expression was observed in NENs from the pancreas (31.5). No pathogenic mutations were significantly associated with ANTXR1H vs L tumors (p&amp;gt;0.05 for all), along with no differences in the prevalence of TMB-H (8.6% vs 12.6%, p=1) or PD-L1+ (5.8%: vs 3.1%, p=1). A greater proportion of B cells (5.34 % vs 3.78%, p&amp;lt;0.001), M1 (1.46% vs 0.15%, p&amp;lt;0.001) and M2 macrophages (3.92% vs 2.67%, p&amp;lt;0.001), and T-regulatory cells (1.77% vs 1.08%, p&amp;lt;0.001) was observed in ANTXR1H TMEs, which were more frequently classified as T cell-inflamed compared to ANTXR1L (42% vs 8%, p&amp;lt;0.001). ANTXR1H tumors also had higher MPAS compared to ANTXR1L (1.16 vs -1.61 arbitrary units, p&amp;lt;0.001). There was no significant difference in median OS between ANTXR1H vs L tumors (18.8 mo. vs 21.2 months, Hazard Ratio:1.18, p=0.114). Conclusion: The increased immune cell infiltrate and prevalence of T-cell inflamed status among ANTXR1H NENs suggests these tumors might be more responsive to treatment with ICIs. As trials incorporating intratumoral injections of SVV-01 in combination with ICIs are underway, further investigation of clinical and molecular associations with ANTXR1 expression in NENs is warranted. Citation Format: Samuel A. Kareff, Harris Krause, Andrew Elliott, Peter Hosein, Emil Lou, Heloisa Soares, Matthew Oberley, Aman Chauhan. The genomic, transcriptomic, and immunologic landscape of TEM8 (ANTXR1) in neuroendocrine neoplasms (NENs) [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 6851.

Artificial Vasa‐Vasorum Serves as an On‐Site Regenerative Promoter of Cell‐Free Vascular Grafting

AbstractThe control paradigm of small vessel pathophysiology has changed to focus on the vascular out‐wall rather than the lumen‐intimal factors. As an emerging controller of the external wall, the microvasculature (“vasa vasorum”) provides interactional routes between the in‐and out‐sides of the vascular wall. Despite numerous approaches to developing small‐diameter vascular grafts, engineering artificial vasa vasorum (AVV) has not been projected as a multi‐functional solution to address long‐standing issues such as thrombotic and immune controls for wall regeneration. Here, the AVV is engineered using a microchannel network hydrogel after a multi‐study validation of implantation functions and then used to wrap the external wall of the cell‐free vessel post‐decellularization while preserving its mechanical properties. Upon inter‐positional and bypass grafting to rabbit arteries, the AVV graft facilitates the recruitment of vascular cells into the cell‐free wall by promoting invasion of angiogenic and vasculogenic cells through the microchannel‐mediated M2 polarization of macrophages. This function results in the efficient restoration of smooth muscle cells, and the revitalized vascular elasticity helps to maintain long‐term patency. The AVV, therefore, serves as an effective catalyst for vascular wall regeneration, offering a solution to clinically successful small‐diameter vascular grafting.