Endothelial Cell Proliferation Research Articles

Dynamically Cross-Linked Double-Network Hydrogels with Matched Mechanical Properties and Ideal Biocompatibility for Artificial Blood Vessels.

Vessel transplantation is currently considered the "gold standard" treatment for cardiovascular disease. However, ideal artificial vascular grafts should possess good biocompatibility and mechanical strength that match those of native autologous vascular tissue to promote in vivo tissue regeneration. In this study, a series of dynamic cross-linking double-network hydrogels and the resultant hydrogel tubes were prepared. The hydrogels (named PCO), composed of rigid poly(vinyl alcohol) (PVA), flexible carboxymethyl chitosan (CMCS), and a cross-linker of aldehyde-based β-cyclodextrin (OCD), were formed in a double-network structure with multiple dynamical cross-linking including dynamic imine bonds, hydrogen bonds, and microcrystalline regions. The PCO hydrogels exhibited superior mechanical strength, good network stability, and fatigue resistance. Additionally, it demonstrated excellent cell and blood compatibility. The results showed that the introduction of CMCS/OCD led to a significant increase in the proliferation rate of endothelial cells seeded on the surface of the hydrogel. The hemolysis rate in the test was lower than 0.3%, and both protein adsorption and platelet adhesion were reduced, indicating an excellent anticoagulant function. The plasma recalcification time test results showed that endogenous coagulation was alleviated to some extent. When formed into blood vessels and incubated with blood, no thrombus formation was observed, and there was minimal red blood cell aggregation. Therefore, this novel hydrogel tube, with excellent mechanical properties, exhibits antiadhesive characteristics toward blood cells and proteins, as well as antithrombotic properties, making it hold tremendous potential for applications in the biomedical and engineering fields.

Nerolidol Alleviates Oxidative Stress and Inflammation in Diabetic Retinopathy Mediated by High Glucose Through Ameliorating Nrf2/HO-1 Pathway

Background Diabetic retinopathy (DR) is the foremost microvascular problem that causes drastic visual impairment in diabetes patients. Hyperglycemia-triggered reaction cascade of inflammation and oxidative stress constitute the DR pathogenesis. The existing treatment options are not completely satisfactory. Materials and Methods We investigated the cell viability by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, inflammatory mediators, lactate dehydrogenase (LDH), superoxide dismutase, glutathione, and malonaldehyde (MDA) levels by ELISA and qRT-PCR assay, protein expression of Nrf2 and heme oxygenase-1 (HO-1) by western blotting assay were analyzed. Results According to our research, nerolidol (NRD) increases the proliferation and antioxidant activity of human retinal endothelial cells (HRECs) by inducing Nrf2/HO-1 signaling, while attenuating MDA, an oxidative stress marker, LDH, and inflammatory mediators. These outcomes suggest that a substantial reaction of inflammation and oxidative stress injury happened in DR, which might be correlated to the instigation of the signaling Nrf2/HO-1. Conclusion NRD effectively suppresses oxidative stress and inflammation in HG-induced HRECs. The primary mechanism of NRD on DR may be linked to the activation of the Nrf2/HO-1 pathway and may give a useful medicine for DR treatment.

Angpt1 binding to Tie1 regulates the signaling required for lymphatic vessel development in zebrafish.

Development of the vascular system is regulated by multiple signaling pathways mediated by receptor tyrosine kinases (RTKs). Among them, angiopoietin (Ang)/Tie signaling regulates lymphatic and blood vessel development in mammals. Of the two Tie receptors, Tie2 is well known as a key mediator of Ang/Tie signaling, but unexpectedly, recent studies reveal that the Tie2 locus has been lost in many vertebrate species, while the Tie1 gene is more commonly present. However, Tie1-driven signaling pathways, including ligands and cellular functions, are not well understood. Here, we performed comprehensive mutant analyses of angiopoietins and Tie receptors in zebrafish and found that only angpt1 and tie1 mutants show defects in trunk lymphatic vessel development. Among zebrafish angiopoietins, only Angpt1 binds to Tie1 as a ligand. We indirectly monitored Ang1/Tie1 signaling and detected Tie1 activation in sprouting endothelial cells (ECs), where Tie1 inhibits nuclear import of EGFP-Foxo1a. Angpt1/Tie1 signaling functions in EC migration, proliferation, and lymphatic specification during early lymphangiogenesis, at least in part by modulating Vegfc/Vegfr3 signaling. Thus, we show Angpt1/Tie1 signaling to constitute an essential signaling pathway for lymphatic development in zebrafish.

Hypoxic adipose stem cell-derived exosomes carrying high-abundant USP22 facilitate cutaneous wound healing through stabilizing HIF-1α and upregulating lncRNA H19.

Hypoxic preconditioning has been recognized as a promotive factor for accelerating cutaneous wound healing. Our previous study uncovered that exosomal lncRNA H19, derived from adipose-derived stem cells (ADSCs), plays a crucial role in orchestrating cutaneous wound healing. Herein, we aimed to explore whether there is a connection between hypoxia and ADSC-derived exosomes (ADSCs-exos) in cutaneous wound healing. Exosomes extracted from ADSCs under normoxic and hypoxic conditions were identified using transmission electron microscope (TEM) and particle size analysis. The effects of ADSCs-exos on the proliferation, migration, and angiogenesis of human umbilical vein endothelial cells (HUVECs) were evaluated by CCK-8, EdU, wound healing, and tube formation assays. Expression patterns of H19, HIF-1α, and USP22 were measured. Co-immunoprecipitation, chromatin immunoprecipitation, ubiquitination, and luciferase reporter assays were conducted to confirm the USP22/HIF-1α/H19 axis, which was further validated in a mice model of skin wound. Exosomes extracted from hypoxia-treated ADSCs (termed as H-ADSCs-exos) significantly increased cell proliferation, migration, and angiogenesis in H2O2-exposed HUVECs, and promoted cutaneous wound healing invivo. Moreover, H-ADSCs and H-ADSCs-exos, which exhibited higher levels of H19, were found to be transcriptionally activated by HIF-1α. Mechanically, H-ADSCs carrying USP22 accounted for deubiquitinating and stabilizing HIF-1α. Additionally, H-ADSCs-exos improved cell proliferation, migration, and angiogenesis in H2O2-triggered HUVECs by activating USP22/HIF-1α axis and promoting H19 expression, which may provide a new clue for the clinical treatment of cutaneous wound healing.

Choline metabolism reprogramming mediates an immunosuppressive microenvironment in non-small cell lung cancer (NSCLC) by promoting tumor-associated macrophage functional polarization and endothelial cell proliferation

IntroductionLung cancer is a prevalent malignancy globally, and immunotherapy has revolutionized its treatment. However, resistance to immunotherapy remains a challenge. Abnormal cholinesterase (ChE) activity and choline metabolism are associated with tumor oncogenesis, progression, and poor prognosis in multiple cancers. Yet, the precise mechanism underlying the relationship between ChE, choline metabolism and tumor immune microenvironment in lung cancer, and the response and resistance of immunotherapy still unclear.MethodsFirstly, 277 advanced non-small cell lung cancer (NSCLC) patients receiving first-line immunotherapy in Sun Yat-sen University Cancer Center were enrolled in the study. Pretreatment and the alteration of ChE after 2 courses of immunotherapy and survival outcomes were collected. Kaplan–Meier survival and cox regression analysis were performed, and nomogram was conducted to identify the prognostic and predicted values. Secondly, choline metabolism-related genes were screened using Cox regression, and a prognostic model was constructed. Functional enrichment analysis and immune microenvironment analysis were also conducted. Lastly, to gain further insights into potential mechanisms, single-cell analysis was performed.ResultsFirstly, baseline high level ChE and the elevation of ChE after immunotherapy were significantly associated with better survival outcomes for advanced NSCLC. Constructed nomogram based on the significant variables from the multivariate Cox analysis performed well in discrimination and calibration. Secondly, 4 choline metabolism-related genes (MTHFD1, PDGFB, PIK3R3, CHKB) were screened and developed a risk signature that was found to be related to a poorer prognosis. Further analysis revealed that the choline metabolism-related genes signature was associated with immunosuppressive tumor microenvironment, immune escape and metabolic reprogramming. scRNA-seq showed that MTHFD1 was specifically distributed in tumor-associated macrophages (TAMs), mediating the differentiation and immunosuppressive functions of macrophages, which may potentially impact endothelial cell proliferation and tumor angiogenesis.ConclusionOur study highlights the discovery of ChE as a prognostic marker in advanced NSCLC, suggesting its potential for identifying patients who may benefit from immunotherapy. Additionally, we developed a prognostic signature based on choline metabolism-related genes, revealing the correlation with the immunosuppressive microenvironment and uncovering the role of MTHFD1 in macrophage differentiation and endothelial cell proliferation, providing insights into the intricate workings of choline metabolism in NSCLC pathogenesis.

Picein alleviates oxidative stress and promotes bone regeneration in osteoporotic bone defect by inhibiting ferroptosis via Nrf2/HO-1/GPX4 pathway.

Osteoporosis (OP) can result in slower bone regeneration than the normal condition due to abnormal oxidative stress and high levels of reactive oxygen species (ROS), a condition detrimental for bone formation, making the OP-related bone healing a significant clinical challenge. As the osteogenic differentiation ability of bone marrow mesenchymal stem cells (BMSCs) is closely related to bone regeneration; currently, this study assessed the effects of Picein on BMSCs in vitro and bone regeneration in osteoporotic bone defect in vivo. Cell viability was determined by CCK-8 assay. The production of (ROS), malonaldehyde, superoxide dismutase activities, and glutathione was evaluated by using commercially available kits, and a flow cytometry analysis was adopted to detect macrophage polarization. Osteogenic capacity of BMSCs was evaluated by alkaline phosphatase (ALP) activity, ALP staining, and Alizarin red S staining. The expression of osteogenic-related proteins (OPN, Runx-2, OCN) and osteogenic-related genes (ALP, BMP-4, COL-1, and Osterix) were evaluated by Western blotting and real-time PCR (RT-PCR). In addition, proliferation, migration ability, and angiogenic capacity of human umbilical vein endothelial cells (HUVECs) were evaluated by EdU staining, scratch test, transwell assay, and tube formation assay, respectively. Angiogenic-related genes (VEGF, vWF, CD31) were also evaluated by RT-PCR. Results showed that Picein alleviated erastin-induced oxidative stress, enhanced osteogenic differentiation capacity of BMSCs, angiogenesis of HUVECs, and protects cells against ferroptosis through Nrf2/HO-1/GPX4 axis. Moreover, Picein regulate immune microenvironment by promoting the polarization of M2 macrophages in vitro. In addition, Picein also reduce the inflammation levels and promotes bone regeneration in osteoporotic bone defect in OP rat models in vivo. Altogether, these results suggested that Picein can promote bone regeneration and alleviate oxidative stress via Nrf2/HO-1/GPX4 pathway, offering Picein as a novel antioxidant agent for treating osteoporotic bone defect.

Effect of High Magnesium and Astragaloside IV on Vascular Endothelial Cells

Effect of High Magnesium and Astragaloside IV on Vascular Endothelial Cells

In vitro studies on the effects of cryopreserved platelet-rich plasma on cells related to wound healing

Platelet-rich plasma (PRP) holds promise as a therapeutic modality for wound healing; however, immediate utilization encounters challenges related to volume, concentration, and consistency. Cryopreservation emerges as a viable solution, preserving PRP’s bioactive components and extending its shelf life. This study explores the practicality and efficacy of cryopreserved platelet-rich plasma (cPRP) in wound healing, scrutinizing both cellular mechanisms and clinical implications. Fresh PRP and cPRP post freeze-thaw underwent assessment in macrophage, fibroblast, and endothelial cell cultures. The impact of cPRP on active component release and cell behavior pertinent to wound healing was evaluated. Varied concentrations of cPRP (1%, 5%, 10%) were examined for their influence on cell polarization, migration, and proliferation. The results showed minimal changes in cPRP’s IL-1β levels, a slight decrease in PDGF-BB, and superior effects on macrophage M2 polarization and fibroblast migration, while no statistical significance was observed in endothelial cell angiogenesis and proliferation. Remarkably, 5% PRP exhibited the most significant stimulation among all cPRP concentrations, notably impacting cell proliferation, angiogenesis, and migration. The discussion underscores that cPRP maintains platelet phenotype and function over extended periods, with 5% cPRP offering the most favorable outcomes, providing a pragmatic approach for cold storage to extend post-thaw viability and amplify therapeutic effects.

The heparin-binding domain of VEGF165 directly binds to integrin αvβ3 and VEGFR2/KDR D1: a potential mechanism of negative regulation of VEGF165 signaling by αvβ3

VEGF-A is a key cytokine in tumor angiogenesis and a major therapeutic target for cancer. VEGF165 is the predominant isoform of VEGF-A, and it is the most potent angiogenesis stimulant. VEGFR2/KDR domains 2 and 3 (D2D3) bind to the N-terminal domain (NTD, residues 1–110) of VEGF165. Since removal of the heparin-binding domain (HBD, residues 111–165) markedly reduced the mitogenic activity of the growth factor, it has been proposed that the HBD plays a critical role in the mitogenicity of VEGF165. Here, we report that αvβ3 specifically bound to the isolated VEGF165 HBD but not to VEGF165 NTD. Based on docking simulation and mutagenesis, we identified several critical amino acid residues within the VEGF165 HBD required for αvβ3 binding, i.e., Arg123, Arg124, Lys125, Lys140, Arg145, and Arg149. We discovered that VEGF165 HBD binds to the KDR domain 1 (D1) and identified that Arg123 and Arg124 are critical for KDR D1 binding by mutagenesis, indicating that the KDR D1-binding and αvβ3-binding sites overlap in the HBD. Full-length VEGF165 mutant (R123A/R124A/K125A/K140A/R145A/R149A) defective in αvβ3 and KDR D1 binding failed to induce ERK1/2 phosphorylation, integrin β3 phosphorylation, and KDR phosphorylation and did not support proliferation of endothelial cells, although the mutation did not affect the KDR D2D3 interaction with VEGF165. Since β3-knockout mice are known to show enhanced VEGF165 signaling, we propose that the binding of KDR D1 to the VEGF165 HBD and KDR D2D3 binding to the VEGF165 NTD are critically involved in the potent mitogenicity of VEGF165. We propose that binding competition between KDR and αvβ3 to the VEGF165 HBD endows integrin αvβ3 with regulatory properties to act as a negative regulator of VEGF165 signaling.

Single-nucleus RNA transcriptome profiling reveals murine adipose tissue endothelial cell proliferation gene networks involved in obesity development

Endothelial cells play an important role in the metabolism of adipose tissue (AT). This study aimed to analyze the changes that adipose tissue in AT endothelial cells undergo during the development of obesity, using single-nucleus RNA sequence (snRNA-seq). Mouse paraepididymal AT cells were subjected to snRNA-seq with the 10X Genomics platform. The cell types were then clustered using t-distributed stochastic neighbor embedding and unbiased computational informatics analyses. Protein–protein interactions network was established using the STRING database and visualized using Cytoscape. The dataset was subjected to differential gene enrichment analysis. In total, 21,333 cells acquired from 24 mouse paraepididymal AT samples were analyzed using snRNA-seq. This study identified 18 distinct clusters and annotated macrophages, fibroblasts, epithelial cells, T cells, endothelial cells, stem cells, neutrophil cells, and neutrophil cell types based on representative markers. Cluster 12 was defined as endothelial cells. The proportion of endothelial cells decreased with the development of obesity. Inflammatory factors, such as Vegfa and Prdm16 were upregulated in the medium obesity group but downregulated in the obesity group. Genes, such as Prox1, Erg, Flt4, Kdr, Flt1, and Pecam1 promoted the proliferation of AT endothelial cells and maintained the internal environment of AT. This study established a reference model and general framework for studying the mechanisms, biomarkers, and therapeutic targets of endothelial cell dysfunction-related diseases at the single-cell level.

Retraction: Lycium barbarum polysaccharide induced apoptosis and inhibited proliferation in infantile hemangioma endothelial cells via down-regulation of PI3K/AKT signaling pathway.

Retraction: Lycium barbarum polysaccharide induced apoptosis and inhibited proliferation in infantile hemangioma endothelial cells via down-regulation of PI3K/AKT signaling pathway.

3D Printing of Succulent‐Inspired Microneedle Array for Enhanced Tissue Adhesion and Controllable Drug Release

AbstractControllable and long‐term release remains a great challenge in current drug delivery systems. Benefiting from their efficient drug loading and painless administration, microneedles (MNs) have emerged as a promising platform for transdermal drug delivery, while they often fail to achieve long‐term tissue adhesion and controllable extended drug release. Here, 3D printing of an innovative MN patch is presented with succulent‐inspired responsive microstructures and light‐controllable long‐term release capability. The MN exhibits a reversible shrink‐swell volume change behavior in response to surrounding humidity, which enables sufficient mechanical strength for skin penetration under the shrinkage conditions and efficient long‐term adhesion when swollen in skin tissues. Moreover, the MN patch introduces a controllable long‐term drug release system, achieved through the integration of thiolated heparin (Hep‐SH) for sustained growth factor release and graphene oxide (GO) nanosheets for controlled drug release via near infrared (NIR) laser irradiation. The MN patches with growth factor loading have good biocompatibility and can promote the proliferation, migration, and proangiogenesis of endothelial cells is further demonstrated. Thus, it is believed that such flexible MN patches can be promising candidates for controllable long‐term transdermal drug delivery as well as other related tissue engineering applications.

TM4SF1 is a molecular facilitator that distributes cargo proteins intracellularly in endothelial cells in support of blood vessel formation

AbstractTransmembrane‐4 L‐six family member‐1 (TM4SF1) is an atypical tetraspanin that is highly and selectively expressed in proliferating endothelial cells and plays an essential role in blood vessel development. TM4SF1 forms clusters on the cell surface called TMED (TM4SF1‐enriched microdomains) and recruits other proteins that internalize along with TM4SF1 via microtubules to intracellular locations including the nucleus. We report here that tumor growth and wound healing are inhibited in Tm4sf1‐heterozygous mice. Investigating the mechanisms of TM4SF1 activity, we show that 12 out of 18 signaling molecules examined are recruited to TMED on the surface of cultured human umbilical vein endothelial cells (HUVEC) and internalize along with TMED; notable among them are PLCγ and HDAC6. When TM4SF1 is knocked down in HUVEC, microtubules are heavily acetylated despite normal levels of HDAC6 protein, and, despite normal levels of VEGFR2, are unable to proliferate. Together, our studies indicate that pathological angiogenesis is inhibited when levels of TM4SF1 are reduced as in Tm4sf1‐heterozygous mice; a likely mechanism is that TM4SF1 regulates the intracellular distribution of signaling molecules necessary for endothelial cell proliferation and migration.

Intervention of exogenous VEGF protect brain microvascular endothelial cells from hypoxia-induced injury by regulating PLCγ/RAS/ERK and PI3K/AKT pathways

Ischemic stroke rapidly increases the expression level of vascular endothelial growth factor (VEGF), which promotes neovascularization during hypoxia. However, the effect and mechanism of VEGF intervention on cerebrovascular formation remain unclear.Therefore, our research discussed the protective effect of exogenous VEGF on cells in hypoxia environment in cerebral microvascular endothelial cells, simulating ischemic stroke in hypoxic environment. Firstly, we detected the proliferation and apoptosis of cerebral microvascular endothelial cells under hypoxia environment, as well the expression levels of VEGF-E, vascular endothelial growth factor re-ceptor-2 (VEGFR-2), BCL2, PRKCE and PINK1. Moreover, immunofluorescence and western blotting were used to verify the regulation of exogenous VEGF-E on VEGFR-2 expression in hypoxic or normal oxygen environment. Lastly, we manipulated the concentration of VEGF-E in the culture medium to investigate its impact on phospholipase Cγ1 (PLCγ1)/extracellular signaling regulatory protein kinase (ERK) -1/2 and protein kinase B (AKT) pathways. Additionally, we employed a PLCγ1 inhibitor (U73122) to investigate its impact on proliferation and PLCγ1/ERK pathways.The results show that hypoxia inhibited the proliferation of cerebral microvascular endothelial cells, promoted cell apoptosis, significantly up-regulated the expression of VEGF-E, VEGFR-2, PRKCE and PINK1, but down-regulated the expression of BCL2. Interference from exogenous VEGF-E activated PLCγ1/ERK-1/2 and AKT pathways, promoting cell proliferation and inhibiting apoptosis of hypoxic brain microvascular endothelial cells.In summary, exogenous VEGF-E prevents hypoxia-induced damage to cerebral microvascular endothelial cells by activating the PLCγ1/ERK and AKT pathways. This action inhibits the apoptosis pathway in hypoxic cerebral microvascular endothelial cells, thereby safeguarding the blood-brain barrier and the nervous system.

Ion channel Piezo1 activation aggravates the endothelial dysfunction under a high glucose environment

BackgroundVasculopathy is the most common complication of diabetes. Endothelial cells located in the innermost layer of blood vessels are constantly affected by blood flow or vascular components; thus, their mechanosensitivity plays an important role in mediating vascular regulation. Endothelial damage, one of the main causes of hyperglycemic vascular complications, has been extensively studied. However, the role of mechanosensitive signaling in hyperglycemic endothelial damage remains unclear.MethodsVascular endothelial-specific Piezo1 knockout mice were generated to investigate the effects of Piezo1 on Streptozotocin-induced hyperglycemia and vascular endothelial injury. In vitro activation or knockdown of Piezo1 was performed to evaluate the effects on the proliferation, migration, and tubular function of human umbilical vein endothelial cells in high glucose. Reactive oxygen species production, mitochondrial membrane potential alternations, and oxidative stress-related products were used to assess the extent of oxidative stress damage caused by Piezo1 activation.ResultsOur study found that in VECreERT2;Piezo1flox/flox mice with Piezo1 conditional knockout in vascular endothelial cells, Piezo1 deficiency alleviated streptozotocin-induced hyperglycemia with reduced apoptosis and abscission of thoracic aortic endothelial cells, and decreased the inflammatory response of aortic tissue caused by high glucose. Moreover, the knockout of Piezo1 showed a thinner thoracic aortic wall, reduced tunica media damage, and increased endothelial nitric oxide synthase expression in transgenic mice, indicating the relief of endothelial damage caused by hyperglycemia. We also showed that Piezo1 activation aggravated oxidative stress injury and resulted in severe dysfunction through the Ca2+-induced CaMKII-Nrf2 axis in human umbilical vein endothelial cells. In Piezo1 conditional knockout mice, Piezo1 deficiency partially restored superoxide dismutase activity and reduced malondialdehyde content in the thoracic aorta. Mechanistically, Piezo1 deficiency decreased CaMKII phosphorylation and restored the expression of Nrf2 and its downstream molecules HO-1 and NQO1.ConclusionIn summary, our study revealed that Piezo1 is involved in high glucose-induced oxidative stress injury and aggravated endothelial dysfunction, which have great significance for alleviating endothelial damage caused by hyperglycemia.

Targeted Rapamycin Delivery via Magnetic Nanoparticles to Address Stenosis in a 3D Bioprinted in Vitro Model of Pulmonary Veins.

Vascular cell overgrowth and lumen size reduction in pulmonary vein stenosis (PVS) can result in elevated PV pressure, pulmonary hypertension, cardiac failure, and death. Administration of chemotherapies such as rapamycin have shown promise by inhibiting the vascular cell proliferation; yet clinical success is limited due to complications such as restenosis and off-target effects. The lack of in vitro models to recapitulate the complex pathophysiology of PVS has hindered the identification of disease mechanisms and therapies. This study integrated 3D bioprinting, functional nanoparticles, and perfusion bioreactors to develop a novel in vitro model of PVS. Bioprinted bifurcated PV constructs are seeded with endothelial cells (ECs) and perfused, demonstrating the formation of a uniform and viable endothelium. Computational modeling identified the bifurcation point at high risk of EC overgrowth. Application of an external magnetic field enabled targeting of the rapamycin-loaded superparamagnetic iron oxide nanoparticles at the bifurcation site, leading to a significant reduction in EC proliferation with no adverse side effects. These results establish a 3D bioprinted in vitro model to study PV homeostasis and diseases, offering the potential for increased throughput, tunability, and patient specificity, to test new or more effective therapies for PVS and other vascular diseases.

Deficiency of the Deubiquitinase UCHL1 Attenuates Pulmonary Arterial Hypertension.

The ubiquitin-proteasome system regulates protein degradation and the development of pulmonary arterial hypertension (PAH), but knowledge about the role of deubiquitinating enzymes in this process is limited. UCHL1 (ubiquitin carboxyl-terminal hydrolase 1), a deubiquitinase, has been shown to reduce AKT1 (AKT serine/threonine kinase 1) degradation, resulting in higher levels. Given that AKT1 is pathological in pulmonary hypertension, we hypothesized that UCHL1 deficiency attenuates PAH development by means of reductions in AKT1. Tissues from animal pulmonary hypertension models as well as human pulmonary artery endothelial cells from patients with PAH exhibited increased vascular UCHL1 staining and protein expression. Exposure to LDN57444, a UCHL1-specific inhibitor, reduced human pulmonary artery endothelial cell and smooth muscle cell proliferation. Across 3 preclinical PAH models, LDN57444-exposed animals, Uchl1 knockout rats (Uchl1-/-), and conditional Uchl1 knockout mice (Tie2Cre-Uchl1fl/fl) demonstrated reduced right ventricular hypertrophy, right ventricular systolic pressures, and obliterative vascular remodeling. Lungs and pulmonary artery endothelial cells isolated from Uchl1-/- animals exhibited reduced total and activated Akt with increased ubiquitinated Akt levels. UCHL1-silenced human pulmonary artery endothelial cells displayed reduced lysine(K)63-linked and increased K48-linked AKT1 levels. Supporting experimental data, we found that rs9321, a variant in a GC-enriched region of the UCHL1 gene, is associated with reduced methylation (n=5133), increased UCHL1 gene expression in lungs (n=815), and reduced cardiac index in patients (n=796). In addition, Gadd45α (an established demethylating gene) knockout mice (Gadd45α-/-) exhibited reduced lung vascular UCHL1 and AKT1 expression along with attenuated hypoxic pulmonary hypertension. Our findings suggest that UCHL1 deficiency results in PAH attenuation by means of reduced AKT1, highlighting a novel therapeutic pathway in PAH.

Potency of the Combination of Chitosan and Hydroxyapatite on Angiogenesis and Fibroblast Cell Proliferation in Direct Pulp Capping of Rattus norvegicus.

The aim of this research was to analyze expression of vascular endothelial growth factor (VEGF), blood vessels, and fibroblast cell proliferation in direct pulp capping treatment of Rattus norvegicus using a combination of chitosan and hydroxyapatite paste. The samples were male R. norvegicus strains of Wistar rats, weighing 200 to 250 g and aged between 8 and 16 weeks. The occlusal surface of the molars of R. norvegicus was prepared with class I cavity and then perforated with the tip of an explorer. Sixty male R. Norvegicus rats were divided into the following: control group (KA) in which the cavity was filled with glass ionomer cement; control group (KB), in which the cavity was filled with Ca(OH)2; PA group, in which the cavity was filled with chitosan (CH); PB group, in which the cavity was filled with hydroxyapatite (HA); and PC group, in which the cavity was filled with chitosan and hydroxyapatite (CH-HA). Each group was divided into 3-, 7-, and 14-day observation groups. The mandibular bone of the molar was cut and histopathological examination was performed to analyze the blood vessels and fibroblast cell proliferation. Immunohistochemistry examination was done to examine the expression of VEGF. The data variation was analyzed with One Way Analysis of Variance (ANOVA) test and continued with multiple comparison Least Significant (LSD) test to determine the different pairs of group. Analysis of variance (ANOVA) showed a significant increase in the expression of VEGF, blood vessels, and fibroblast cell proliferations (p ≤ 0.05;), especially in the PC group compared to the other four groups. The least significant test (LSD) test showed significant differences between the groups on the expression of VEGF, blood vessels, and fibroblast cell proliferations. The combination of chitosan and hydroxyapatite could promote healing of direct pulp capping treatment by increasing the expression of VEGF, blood vessel, and fibroblast cell proliferation.

Abstract PO3-25-07: Characterization of tertiary lymphoid structures and its association with response to immune checkpoint inhibitors in triple negative breast cancer using spatial transcriptomics

Abstract Background Tertiary lymphoid structures (TLS) are ectopic lymphoid organs playing a role in adaptive antitumor immune response. They were shown to be associated with favorable outcome and appears to be a promising biomarker for response to immune checkpoint inhibitors (ICI). Yet, there is no consensus for their detection and quantification. Here we aimed to derive a specific TLS signature using high resolution spatial transcriptomics data from a large series of triple negative breast cancer (TNBC) samples and to assess its clinical relevance including response to ICI. Methods Spatial transcriptomics (ST) was performed on a series of 94 early-stage TNBC surgical samples with detailed clinicopathological and outcome data. TLS and tumor infiltrating lymphocytes (TILs) were scored using hematoxylin and eosin and double immunohistochemistry (CD3/CD20) stained slides by a dedicated breast pathologist. Regression based on morphological annotations and deconvolution methods estimated the fraction of gene expression related to TLS in each ST spot allowing to derive specific TLS signature. Its prognostic and predictive value of response to ICI was assessed using publicly available breast cancer (METABRIC, SCAN-B, and I-SPY2) and other tumor type datasets treated with ICI (Bareche et al. Ann Oncol, 2022) from bulk gene expression data. Results Deconvolution of the TLS compartments using the ST gene expression data showed that TLS are enriched in all B cell subsets, CD4+ (central) memory and naïve T cells, as well as mast cells. A functional analysis showed an enrichment of ‘vascular endothelial cell proliferation’, ‘mitotic G2/M transition checkpoint’, ‘V/D/J recombination’ and ‘regulation of cell chemotaxis to fibroblast growth factor’ suggesting that TLS aggregates are associated with angiogenesis, immune cell proliferation, adaptative humoral response and tissue remodeling. A comparison of gene expression data between TLS and non-aggregated TILs led to the development of a specific TLS gene signature comprising B and T cell-specific genes, immunoglobulin genes as well as genes associated with TLS initiation. Its projection on tumor slides overlapped with the regions annotated as TLS by the pathologist, demonstrating its specificity for TLS detection. As expected, the highest levels of the TLS signature were observed in the immunomodulatory TNBC molecular subtype and fully inflamed tumors, whereas the lowest levels were observed in the mesenchymal molecular subtype and immune desert tumors (p< 0.05). Of note, high levels of the TLS signature were associated with better prognosis in TNBC patients from METABRIC (p=6.5 10−5) and SCAN-B datasets (p=1.6 10−3) as well as with higher pathological complete response rates in all breast cancer patients treated with neoadjuvant pembrolizumab in the I-SPY2 study (p=0.026). Similar results were found in other tumor types treated with immunotherapy in the metastatic setting including metastatic melanoma, pancreatic and bladder cancers (PFS, p=6 10−6). Of interest, the TLS signature outperformed the predictive value of other immune-related signatures including TILs suggesting a key role of TLS in obtaining a sustainable, adaptive antitumor immune response in the vicinity of the tumor area. Conclusion By leveraging the potential of spatial transcriptomics, we have successfully developed a unique TLS gene signature that demonstrates a strong correlation with the response to ICI in breast cancer and various other tumor types. This TLS gene signature outperforms other immune biomarkers, underscoring the pivotal role of TLS in eliciting a robust antitumor immune response. The universality of this TLS signature makes it a potent biomarker capable of identifying patients who would benefit from immunotherapy, pending further validation. Citation Format: Xiaoxiao Wang, David Venet, Frédéric Lifrange, Denis Larsimont, Mattia Rediti, Linnea Stenbeck, Floriane Dupont, Ghizlane Rouas, Andrea Joaquin Garcia, David Gacquer, Ligia Craciun, Laurence Buisseret, Nayanika Bhalla, Yuvarani Masarapu, Kim Thrane, Eva Gracia Villacampa, Lovisa Franzén, Sami Saarenpää, Linda Kvastad, Joakim Lundeberg, Françoise Rothé, Christos Sotiriou. Characterization of tertiary lymphoid structures and its association with response to immune checkpoint inhibitors in triple negative breast cancer using spatial transcriptomics [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 PO3-25-07.

Designing and synthesis of injectable hydrogel based on carboxymethyl cellulose/carboxymethyl chitosan containing QK peptide for femoral head osteonecrosis healing

Femoral head necrosis is a debilitating disorder that typically caused by impaired blood supply to the hip joint. In this study, a novel injectable hydrogel based on Oxidized Carboxymethyl Cellulose (OCMC)-Carboxymethyl Chitosan (CMCS) polymers containing an angiogenesis stimulator peptide (QK) with a non-toxic crosslinking interaction (Schiff based reaction) was synthesized to enhance angiogenesis following femoral head necrosis in an animal model. The physicochemical features of fabricated injectable hydrogel were analyzed by FTIR, swelling and degradation rate, rheometry, and peptide release. Also, the safety and efficacy were evaluated following an in vitro hydrogel injection study and an avascular necrosis (AVN) animal model. According to the results, the hydrogel exhibited an appropriate swelling ratio and water uptake (>90 %, 24 h) as well as a suitable degradation rate over 21 days accompanied by a continuous peptide release. Also, data showed that hydrogels containing QK peptide boosted the proliferation, differentiation, angiogenesis, and osteogenic potential of both Bone Marrow mesenchymal Stem Cells (BM-MSCs) and human umbilical vein endothelial cells (HUVECs) (****p < 0.0001 and ***p < 0.001, respectively). Furthermore, molecular and histological evaluations significantly demonstrated the overexpression of Runx2, Osteocalcin, Collagen I, VEGF and CD34 genes (**p < 0.01 and ***p < 0.001, respectively), and also femoral head necrosis was effectively prohibited, and more blood vessels were detected in defect area by OCMC-CMCS hydrogel containing QK peptide (bone trabeculae >9000, ***p < 0.001). In conclusion, the findings demonstrate that OCMC-CMCS-QK injectable hydrogel could be considered as an impressive therapeutic construct for femoral head AVN healing.