Endothelial Cell Marker CD31 Research Articles

Linc-ROR Modulates the Endothelial-Mesenchymal Transition of Endothelial Progenitor Cells through the miR-145/Smad3 Signaling Pathway

The endothelial-mesenchymal transition (EndMT) of endothelial progenitor cells (EPCs) plays a notable role in pathological vascular remodeling. Emerging evidence indicated that long non-coding RNA-regulator of reprogramming (linc-ROR) can promote epithelial-mesenchymal transition (EMT) in a variety of cancer cells. Nevertheless, the function of linc-ROR in EPC EndMT has not been well elucidated. The present study investigated the effect and possible mechanisms of function of linc-ROR on the EndMT of EPCs. A linc-ROR overexpression lentiviral vector (LV linc-ROR) or a linc-ROR short hairpin RNA lentiviral vector (LV-shlinc-ROR) was used to up or downregulate linc-ROR expression in EPCs isolated from human umbilical cord blood. Functional experiments demonstrated that LV-linc-ROR promoted the proliferation and migration of EPCs, but inhibited EPC angiogenesis in vitro. In the meantime, reverse transcription-quantitative PCR and western blotting results showed that the expression of the endothelial cell markers vascular endothelial-cadherin and CD31 was decreased, while the expression of the mesenchymal cell markers α-smooth muscle actin and SM22α was increased at both mRNA and protein levels in LV-linc-ROR-treated EPCs, indicating that linc-ROR induced EPC EndMT. Mechanistically, the dual-luciferase reporter assay demonstrated that microRNA (miR/miRNA)-145 was a direct target of linc-ROR, and miR-145 binds to the 3’-untranslated region of Smad3. Moreover, LV-shlinc-ROR increased the expression of miR-145, but decreased the expression of Smad3. In conclusion, linc-ROR promotes EPC EndMT, which may be associated with the miR-145/Smad3 signaling pathway.

Carotid artery transplantation of brain endothelial cells enhances neuroprotection and neurorepair in ischaemic stroke rats.

Brain microvascular endothelial cells (BMECs), an important component of the neurovascular unit, can promote angiogenesis and synaptic formation in ischaemic mice after brain parenchyma transplantation. Since the therapeutic efficacy of cell-based therapies depends on the extent of transplanted cell residence in the target tissue and cell migration ability, the delivery route has become a hot research topic. In this study, we investigated the effects of carotid artery transplantation of BMECs on neuronal injury, neurorepair, and neurological dysfunction in rats after cerebral ischaemic attack. Purified passage 1 endothelial cells (P1-BMECs) were prepared from mouse brain tissue. Adult rats were subjected to transient middle cerebral artery occlusion (MCAO) for 30 min. Then, the rats were treated with 5 × 105 P1-BMECs through carotid artery infusion or tail vein injection. We observed that carotid artery transplantation of BMECs produced more potent neuroprotective effects than caudal injection in MCAO rats, including reducing infarct size and alleviating neurological deficits in behavioural tests. Carotid artery-transplanted BMECs displayed a wider distribution in the ischaemic rat brain. Immunostaining for endothelial progenitor cells and the mature endothelial cell markers CD34 and RECA-1 showed that carotid artery transplantation of BMECs significantly increased angiogenesis. Carotid artery transplantation of BMECs significantly increased the number of surviving neurons, decreased the cerebral infarction volume, and alleviated neurological deficits. In addition, we found that carotid artery transplantation of BMECs significantly enhanced ischaemia-induced hippocampal neurogenesis, as measured by doublecortin (DCX) and Ki67 double staining within 2 weeks after ischaemic injury. We conclude that carotid artery transplantation of BMECs can promote cerebral angiogenesis, neurogenesis, and neurological function recovery in adult rats after ischaemic stroke. Our results suggest that carotid injection of BMECs may be a promising new approach for treating acute brain injuries.

Intraluminal release of citrullinated histone 3 from various cellular origins coincides with microvascular thrombosis in burn wounds.

Loss of perfusion in the burn wound might cause wound deepening and impaired healing. We previously showed persistent microvascular thrombosis coinciding with intraluminal neutrophils extracellular traps in human burned skin. This study investigates the presence of intraluminal citrullinated histone 3 (H3cit) from different cellular origins (neutrophils, monocytes, and lymphocytes) in relation to microvascular thrombosis of burn wounds. Eschar was obtained from burn patients (n = 18) 6-40 days postburn with a mean total burned body surface area of 23%. Microvascular presence of tissue factor (TF), factor XII (FXII) and thrombi was assessed by immunohistochemistry. Intramicrovascular cell death was analyzed via immunofluorescent microscopy, combining antibodies for neutrophils (MPO), monocytes (CD14), and lymphocytes (CD45) with endothelial cell markers CD31 and H3cit. Significantly increased microvascular expression of TF, FXII, and thrombi (CD31+) was found in all eschar samples compared with control uninjured skin. Release of H3cit from different cellular origins was observed in the lumen of the dermal microvasculature in the eschar tissue 7-40 days postburn, with release from neutrophilic origin being 2.7 times more abundant. Intraluminal presence of extracellular H3cit colocalizing with either MPO, CD14, or CD45 is correlated to increased microvascular thrombosis in eschar of burn patients.

A novel approach for transforming breast cancer stem cells into endothelial cells.

Tumor vascular endothelial cells play a pivotal in the tumor microenvironment, influencing the proliferation, invasion, and metastasis of tumor progression. The present study investigated a novel method for inducing the transformation of breast cancer stem cells into endothelial cells, providing a cellular model investigating anti-angiogenic mechanisms in vitro. The breast cancer cell line MCF-7 was used, and the expression of CD133 was initially detected using flow cytometry. CD133+ breast cancer cells were purified using immunomagnetic bead sorting technology, yielding an MCF-7CD133+ subpopulation. The proliferation ability of these cells was assessed using an MTT assay, while their microsphere formation ability was evaluated using a microsphere formation assay. Post-transformation in an optimized endothelial cell culture medium, expression of endothelial cell markers CD31 and CD105 were detected using flow cytometry. Endothelial cell tube formation assays and DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) assays were employed to analyze the endothelial cell function of the MCF-7CD133+ cells. MDM2/CEN12 gene amplification was detected through fluorescence in situ hybridization (FISH). The MCF-7 breast cancer cell line exhibited 1.7±0.3% trace cells expressing the stem cell surface marker CD133. After anti-CD133 immunomagnetic bead sorting, MCF-7CD133+ and MCF-7CD133- subpopulation cells were obtained, with CD133 expression rates of 85.6±2.8 and 0.18±0.08%, respectively. MTT assay results demonstrated that, after 7 days, the proliferation rate of MCF-7CD133+ cells was significantly higher compared with MCF-7CD133- cells. MCF-7CD133+ subpopulation cells displayed strong stem cell characteristics, growing in suspension in serum-free media and forming tumor cell spheres. In contrast, MCF-7CD133- cells failed to form microspheres. After culturing cells in endothelial cell differentiation and maintenance media, the percentage of MCF-7CD133+ cells before and after endothelial cell culture was 0.3±0.16 and 81.4±8.37% for CD31+ cells and 0.2±0.08 and 83.8±7.24% for CD105+ cells, respectively. Vascular-like structure formation and Ac-LDL phagocytosis with red fluorescence in the tube formation assays confirmed endothelial cell function in the MCF-7CD133+ cells. FISH was used to verify MDM2/CEN12 gene amplification in the induced MCF-7CD133+ cells, indicating tumor cell characteristics. The modified endothelial cell transformation medium effectively induced differentiated tumor stem cells to express vascular endothelial cell markers and exhibit endothelial functions, ideal for in vitro anti-angiogenesis research.

Capturing Endothelial-to-Hematopoietic Transition Using a Highly Efficient in Vitro Platform

Introduction De novo generation of hematopoietic stem and progenitor cells (HSPCs) for therapeutic use has been a long-standing goal of regenerative medicine. During development, the first definitive hematopoietic stem cells (HSCs) derive from a small number of specialized vascular endothelial cells (EC), named hemogenic endothelial cells (HECs), located in the Aorta-Gonado-Mesonephrose (AGM). The process of HEC conversion to HSPCs is known as endothelial-to-hematopoietic transition (EHT). During EHT, endothelial features are lost while switching on hematopoietic transcriptional programs leading to drastic functional and morphological changes that generate free-floating, round hematopoietic cells from large, flat, anchorage-dependent ECs. Key steps regulating EHT are difficult to elucidate using in vivo models because the hemogenic capacity of HECs occurs within a 2-day developmental window. Only a tiny number of cells undergo EHT in vivo. New model systems that overcome current in vivo obstacles are required so the details of EHT and the mechanisms that lead to the generation of HSPCs can be better understood. We previously reported that fully-committed ECs can be converted to functional HSPCs by ectopic expression of transcription factors Spi1, FOSB, GFI1, and Runx1 (SFGR) and an appropriate vascular niche (Sandler et al. Nature, 2014; Lis et al. Nature, 2017). But conversion using this method required several weeks and was associated with variable and low efficiency. Here, we show that our new in vitro EHT platform (ivEHT), very closely recapitulates the process of EHT in vivo and can be used to uncover heretofore unrecognized steps necessary for EC conversion to HSPCs. Methods All steps in the in vitro conversion of ECs to HSPCs were systematically analyzed and optimized. The refined model utilized new media constituents and polycistronic constructs to ensure ectopic expression of transcription factors Spi1, FOSB, GFI1 and Runx1 (SFGR)in human umbilical vein-ECs (HUVECs) in appropriate ratios and sequence. Results The new ivEHT platform yields efficient EC conversion to hematopoietic like CD45+ cells (≥80%) and high temporal reproducibility of conversion that is largely complete within 96 hrs. The ivEHT sequence of conversion closely follows the timing and sequence of EHT in vivo. Initially, transduced HUVECs are large, flat cells with CDH5-marked intercellular junctions and firm adhesion to basement membrane. Within the first 24hrs of induction, SFGR-HUVECs downregulate ECs markers CD31, KDR, CDH5 and activate the endogenous RUNX1 locus detected by the expression of nuclear GFP (GFPn) under control of the human RUNX1+24 enhancer (detected using a lentiviral reporter vector). Day 2 (D2) is characterized by complete clearance of endothelial-specific Weibel-Palade Bodies (WPBs, marked by Von Willebrand Factor, VWF) and excess mitochondria. This window also initiates morphologic changes in RUNX1-GFPn marked cells with loss of intercellular junctions and attachment to basement membrane and initial expression of hematopoietic markers such as CD45. Days 3-4 are marked by loss of cell volume and cytoplasm, cellular rounding and detachment from matrix, and increased expression of CD45 and other hematopoietic markers like cKIT, MPL, CD43. These stages of conversion are only identified in RUNX1-GFPn marked cells and by the end of D5, ~80% of cells have fully converted to CD45+ cells. Conversion is not accompanied by cell proliferation, but after D5 cell numbers expand rapidly. The ivEHT platform is scalable and highly reproducible making it amenable to higher-throughput testing in 96-well format using flow cytometry readouts. Using the ivEHT platform, we have captured and validated in AGM explants previously unobserved aspects of early EHT that is complete between D2-D3. Discussion This work promises a reproducible, faithful, and scalable platform which generates hematopoietic like cells while successfully recapitulating early steps of the EHT in the AGM. Importantly, this platform is readily accessible and is easily customized for discovery of EHT molecular details currently hidden to science, allowing significant advances in the understanding of how HSPCs emergence in vivo.

SGLT2 expression in the left ventricle of cardiac patients is correlated with low-grade inflammation involving the pro-oxidant AT1R/NADPH oxidases/SGLT2 crosstalk: potential role in heart failure

Abstract Introduction Clinical studies showed that sodium-glucose co-transporter2 inhibitors (SGLT2i) have beneficial effects in heart failure patients regardless of ejection fraction and diabetes. Such cardiac diseases are often characterized by low-grade inflammation and impaired endothelial coronary microcirculation function. Recently, angiotensin II and TNF-α were shown to induce SGLT2 expression in endothelial cells (ECs) to sustain oxidative stress leading to endothelial dysfunction. However, the role and function of SGLT2 in the human left ventricle (LV) remain unclear. Therefore, this study evaluated the expression of SGLT2 in the LV of patients with cardiac diseases and, if so, determined the cellular localization, the underlying mechanism and the functional role. Methods Human LV biopsies were collected from 20 patients subjected to valve surgery at the Nouvel Hôpital Civil, Strasbourg, France. Expression levels of targets were determined by RT-qPCR and Western blot analysis, the in situ tissue localization by immunofluorescence (IF) staining and oxidative stress by dihydroethidium staining. Results RT-qPCR analysis of LV revealed variable SGLT1 and SGLT2 mRNA levels with no significant correlation. However, SGLT2 mRNA expression showed a positive correlation with those of pro-inflammatory markers (IL-1ß, IL-6, TNF-α, MCP-1 and CD68), AT1R, and VCAM-1. Western blot analysis confirmed the heterogenous SGLT2 protein expression level in LV with a difference up to 3.5-fold among the specimens. SGLT2 protein levels showed a positive correlation with markers of oxidative stress (phospho-p65 NF-κB, AT1R, nitrotyrosine) and markers of ECs activation (VCAM-1, ICAM-1), and a negative correlation with levels of eNOS. Further, immunofluorescence analysis of LV cryosections showed SGLT2 staining in the endothelium of the coronary microcirculation, as indicated by its co-localization with the endothelial cell marker CD31, and throughout the heart tissue. SGLT2 signals were also observed in areas showing IF signals for TNF-α, phospho-p65 NF-κB, CD68 and VCAM-1 suggesting that SGLT2 expression is localized to sites of inflammation. LV specimens with high SGLT2 protein levels showed elevated levels of oxidative stress, which were inhibited by N-acetylcysteine, VAS-2870, perindoprilat, losartan, empagliflozin, and infliximab. Conclusions The findings indicate that endothelial and cardiac SGLT2 expression in the LV of cardiac patients is associated with low-grade inflammation and oxidative stress. They further indicate that the pro-inflammatory cytokines/AT1R/NAPDH oxidases/SGLT2 crosstalk contributes to sustain a pro-oxidant state. Thus, SGLT2 inhibitors might ameliorate HF by mitigating the low-grade inflammatory-related pro-oxidant state and, hence, preserve the crucial endothelial control of the cardiomyocyte function in the LV.

P12.10.A VASCULOGENIC MIMICRY OCCURS IN PRIMARY AND RECURRENT GLIOBLASTOMA AND IS NOT ASSOCIATED WITH PSMA EXPRESSION

Abstract BACKGROUND Glioblastomas are highly vascular tumours that overexpress vascular endothelial growth factor (VEGF), however the anti-VEGF antibody bevacizumab has not demonstrated significant overall survival benefit for patients. Vasculogenic mimicry (VM), the ability of tumour cells to form functional microvasculature lacking an endothelial lining, may compensate for insufficient or inhibited angiogenesis. VM has been reported in primary glioblastoma tissue, but not investigated at progression in recurrent tumours. Prostate specific membrane antigen (PSMA) is expressed in abnormal vessels within primary glioblastoma tumours. We aimed to determine whether VM occurs in primary and recurrent glioblastoma tumours and whether VM vessels also express PSMA. MATERIAL AND METHODS Thirty-five matched pairs of primary and recurrent formalin-fixed paraffin-embedded glioblastoma tissue were immunohistochemically labelled for the endothelial cell marker CD34 and PSMA, and counterstained with periodic acid-Schiff (PAS). Vascular structures were categorised as endothelial vessels (CD34+/PAS+) or VM (CD34-/PAS+). Vessels were counted in ten regions within each tumour and a vessel density/mm2 for each vessel category was determined. RESULTS Data are expressed as median values. Endothelial vessels accounted for most blood vessels observed. There was a significant decrease in endothelial vessel density in recurrent (41.34 vessels/mm2) compared to primary (86.98 vessels/mm2) glioblastomas (p <0.001). VM was observed in 15/35 primary tumours (42.86%) and 10/35 recurrent tumours (28.57%). VM accounted for only a small proportion of the overall vasculature and VM density was not different between the primary and recurrent groups (p = 0.266). Kaplan-Meier analyses with log rank tests indicated that VM-tumours had longer median OS time than VM+ tumours, regardless of whether VM status was determined based on the primary or recurrent tumour. However, there was no significant difference in survival distribution between VM+ and VM-tumours (primary χ2(1) = 0.381, p = 0.537; recurrent χ2(1) = 3.552, p = 0.059). PSMA is expressed in endothelia lined vessels based on serial section labelling for PSMA and CD34. PSMA expression significantly decreased in recurrent (median H-score = 0.22) compared to primary (median H-score = 0.44) tumours (one-tailed p = 0.031; n = 32) mirroring the CD34+ vessel density. CONCLUSION VM contributes to a relatively small proportion of the overall vasculature in glioblastoma tumours and was not associated with OS nor the expression of PMSA. Since VM- tumours had longer OS than VM+ tumours, assessment of VM in recurrent tumours in a larger cohort may determine if there is a clinical significance for VM in recurrent glioblastoma.

A method to isolate human dermal microvascular pericytes without the use of magnetic beads sorting in vitro

Human dermal microvascular pericytes (HDMPCs) are a critical component of the skin flap microvasculature and play a role in regulating flap blood flow and integrity. Pericytes were isolated mostly via magnetic bead sorting in the published literature. In this study, we discuss in detail how to separate and concentrate pericytes from human facial flaps using enzyme digestion and differential adherence instead of magnetic bead sorting. Cultured HDMPCs were seen to have well-spread irregular edges, with most cells having two longitudinal pericytic processes. The phalloidin staining revealed that HDMPCs had prominent stress fibers, and the nucleus deviated to the side that interacted with the neighboring pericytic processes. Flow cytometry analysis showed that the positive rates of NG2 in the first and second passages were 91.2% ± 0.7% and 98.2% ± 0.1% separately. And the immunofluorescence and western blot results demonstrated a positive expression of α smooth muscle actin (αSMA), platelet-derived growth factor receptor β (PDGFRβ), and NG-2, while the endothelial cell marker CD31 was negatively expressed. In summary, we established a straightforward methodology for selectively isolating and identifying HDMPCs as well as generating high-purity cell cultures in vitro without the use of magnetic bead sorting.

Vessel size as a marker of survival in estrogen receptor positive breast cancer

PurposeAngiogenesis is crucial for tumor growth and is one of the hallmarks of cancer. In this study, we analyzed microvessel density, vessel median size, and perivascular a-SMA expression as prognostic biomarkers in breast cancer.MethodsDual IHC staining was performed where alpha-SMA antibodies were used together with antibodies against the endothelial cell marker CD34. Digital images of stainings were analyzed to extract quantitative data on vessel density, vessel size, and perivascular alpha-SMA status.ResultsThe analyses in the discovery cohort (n = 108) revealed a statistically significant relationship between large vessel size and shorter disease-specific survival (p = 0.007, log-rank test; p = 0.01, HR 3.1; 95% CI 1.3–7.4, Cox-regression analyses). Subset analyses indicated that the survival association of vessel size was strengthened in ER + breast cancer. To consolidate these findings, additional analyses were performed on a validation cohort (n = 267) where an association between large vessel size and reduced survival was also detected in ER + breast cancer (p = 0.016, log-rank test; p = 0.02; HR 2.3, 95% CI 1.1–4.7, Cox-regression analyses).ConclusionAlpha-SMA/CD34 dual-IHC staining revealed breast cancer heterogeneity regarding vessel size, vessel density, and perivascular a-SMA status. Large vessel size was linked to shorter survival in ER + breast cancer.

Human Saphenous Vein Endothelial Cell Isolation and Exposure to Controlled Levels of Shear Stress and Stretch.

Coronary artery bypass graft (CABG) surgery is a procedure to revascularize ischemic myocardium. Saphenous vein remains used as a CABG conduit despite the reduced long-term patency compared to arterial conduits. The abrupt increase of hemodynamic stress associated with the graft arterialization results in vascular damage, especially the endothelium, that may influence the low patency of the saphenous vein graft (SVG). Here, we describe the isolation, characterization, and expansion of human saphenous vein endothelial cells (hSVECs). Cells isolated by collagenase digestion display the typical cobblestone morphology and express endothelial cell markers CD31 and VE-cadherin. To assess the mechanical stress influence, protocols were used in this study to investigate the two main physical stimuli, shear stress and stretch, on arterialized SVGs. hSVECs are cultured in a parallel plate flow chamber to produce shear stress, showing alignment in the direction of the flow and increased expression of KLF2, KLF4, and NOS3. hSVECs can also be cultured in a silicon membrane that allows controlled cellular stretch mimicking venous (low) and arterial (high) stretch. Endothelial cells' F-actin pattern and nitric oxide (NO) secretion are modulated accordingly by the arterial stretch. In summary, we present a detailed method to isolate hSVECs to study the influence of hemodynamic mechanical stress on an endothelial phenotype.

Abstract 3665: Umbilical cord blood endothelial progenitor cell-derived extracellular vesicles control important endothelial cell functions

Abstract Background: Circulating endothelial progenitor cells (EPCs) play a pivotal role in vascular regeneration and the repair of diseases in which angiogenesis is required, including ischemia and tissue transplantation. Although they are a potentially valuable cell therapy tool, their clinical use remains limited due to suboptimal storage conditions and especially, immune rejection. Even if these cells have been shown to be tolerated by the immune system, their in vivo long-term effects still remain to be elucidated. EPC-derived extracellular vesicles (EPC-EVs) may be an attractive alternative to EPCs given their key role in cell-cell communication and expression of the same parental markers. In this study, we investigated the regenerative effects of umbilical cord blood EPC-EVs on EPCs in vitro. Methods: EPCs were obtained from human umbilical cord blood (CB) and characterized by flow cytometry. After amplification, EPCs were cultured in a medium containing an EVs-depleted serum (EV-free medium). Then the conditioned medium was used to isolate the EVs with Tangential Flow Filtration (TFF). The EVs were characterized by a series of tests defining their size, number, and protein content. The regenerative effects of EPC-EVs on endothelial cells were investigated by analyzing cell migration, wound healing, and tube formation. We also analyzed the effect of EPC-EVs on endothelial cell inflammation and NO production. Results: We showed that adding different doses of EPC-EVs on EPCs does not alter the basal expression of the endothelial cell markers CD31, CD144, and KDR, nor change their proliferative potential and NO production level. Furthermore, we demonstrated that EPC-EVs, when used at a higher than physiologic dose, create a mild inflammatory condition that activates EPCs and boosts their regenerative features like migration and tube formation functions. Conclusions: Our results reveal for the first time that EPC-EVs, when used at a high dose, could enhance EPC regenerative functions without altering their endothelial identity. Citation Format: Sawssen Ben Fraj, Sina Naserian, Bileyle Lorenzini, Sylvie Goulinet, Philippe Mauduit, Georges Uzan, Houda Haouas. Umbilical cord blood endothelial progenitor cell-derived extracellular vesicles control important endothelial cell functions. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3665.

Thymic cavernous haemangioma in a dog

A 12-year-old male neutered Australian Shepherd Dog was presented to Charlotte Animal Referral & Emergency with a history of a thoracic mass. On physical examination, physiological parameters were within the normal ranges, and a complete haemogram and serum biochemistry profile were unremarkable except for mild thrombocytopenia. A computed tomography scan revealed a 21 × 15 × 12.7 cm thoracic mass encompassing the cranial mediastinum and extending to the right caudal thorax. The mass was surgically removed and histopathological evaluation revealed that it comprised remnants of the thymus and a neoplasm composed of large blood-filled vascular spaces lined by a single layer of endothelial cells with mild anisocytosis and anisokaryosis. The neoplastic cells had diffuse strong immunolabeling for endothelial cell marker CD31. Multifocally, there were large cystic degenerated areas of thymic tissue lined by plump cytokeratin AE1/AE3-positive epithelial cells. Based on these findings, a diagnosis of thymic cavernous haemangioma (CH) was made. Thymic CH is rare in animals, with the only reported case in a cross bred cow. To our knowledge, this is the first case of a thymic CH in a dog.

The Effect of N6-Methyladenosine Regulators and m6A Reader YTHDC1-Mediated N6-Methyladenosine Modification Is Involved in Oxidative Stress in Human Aortic Dissection.

Aortic dissection (AD) develops pathological changes in the separation of the true and false aortic lumen, with high lethality. m6A methylation and oxidative stress have also been shown to be involved in the onset of AD. Through bioinformatics methods, three differentially expressed m6A regulators (YTHDC1, YTHDC2, and RBM15) were excavated from the GSE52093 dataset in the Gene Expression Omnibus (GEO) database, and functional enrichment analysis of the differentially expressed genes (DEGs) regulated by m6A regulators was performed. Then, the genes with oxidative stress-related functions among these genes were found. The protein interaction network of the oxidative stress-related genes and the competing endogenous RNA- (ceRNA-) miRNA-mRNA network were constructed. Among them, DHCR24, P4HB, and PDGFRA, which have m6A differences in AD samples, were selected as key genes. We also performed immune infiltration analysis, as well as cell-gene correlation analysis, on samples from the dataset. The results showed that YTHDC1 was positively correlated with macrophage M1 and negatively correlated with macrophage M2. Finally, we extracted AD and healthy aorta RNA and protein from human tissues that were taken from AD patients and patients who received heart transplants, performed quantitative real-time PCR (qRT-PCR) on YTHDC2 and RBM15, and performed qRT-PCR and western blot (WB) detection on YTHDC1 to verify their differences in AD. The mRNA and protein levels of YTHDC1 were consistent with the results of bioinformatics analysis and were downregulated in AD. Immunofluorescence (IF) was used to colocalize YTHDC1 and endothelial cell marker CD31. After knocking down YTHDC1 in human umbilical vein endothelial cells (HUVECs), reactive oxygen species (ROS) levels had a tendency to increase and the expression of peroxide dismutase SOD2 was decreased. This study provides assistance in discovering the role of m6A regulator YTHDC1 in AD. In particular, m6A modification participates in oxidative stress and jointly affects AD.

Sodium alginate/gelatin hydrogels loaded with adipose-derived mesenchymal stem cells promote wound healing in diabetic rats.

Chronic refractory wounds are a common complication in diabetic patients. Adipose-derived mesenchymal stem cells (ASCs) have been shown to play an essential role in diabetic wound repair. To determine whether a composite of ASCs and sodium alginate/gelatin (Gel-Al) hydrogel can promote diabetic wound healing. Full-thickness cutaneous wounds were created in streptozotocin-induced diabetic rats prior to treatment with Gel-Al hydrogels loaded with ASCs. Hydrogel biocompatibility and wound healing were analyzed. Hematoxylin and eosin staining, Masson staining, immunofluorescence, enzyme-linked immunosorbent assays (ELISA), and quantitative real-time PCR were performed for the assessment of cellular responses. Compared to the control group or Gel-Al alone group, the combination of Gel-Al and ASCs promoted wound closure, facilitated granulation tissue regeneration and collagen deposition, and upregulated the expression of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and endothelial cell marker CD31. Moreover, the combination of Gel-Al and ASCs decreased interleukin-6 (IL-6) and interleukin-1β (IL-1β) expression, increased transforming growth factor beta1 (TGFβ1), interleukin-10 (IL-10), interleukin-4 (IL-4) and interleukin-13 (IL-13) expression, and increased M2 macrophage polarization. Gel-Al hydrogels loaded with ASCs accelerate diabetic wound healing. The Gel-Al hydrogel-based ASC system therefore represents an innovative therapeutic strategy for diabetic wound repair.

Antiangiogenic activity of the penicillin derivative TAP7f in melanoma.

Previously , we demonstrated that the non-antibiotic penicillin derivative TAP7f inhibited melanoma metastasis in vitro and in vivo through the downregulation of β-catenin and integrin αVβ3. As angiogenesis is required for tumor growth and metastasis, we decided to explore the possible antiangiogenic effect of TAP7f. We found that TAP7f inhibited proliferation, migration, tube formation, and actin cytoskeleton organization of human endothelial cells. In a gel plug assay, an in vivo model for angiogenesis, TAP7f also blocked vascular formation induced by fibroblast growth factor 2. Furthermore, when murine B16-F10 melanoma cells pre-treated with TAP7f were injected intradermally in mice, we observed a decrease in the number and thickness of the capillaries surrounding the tumor. Additionally, TAP7f downregulated vascular endothelial growth factor (VEGF) and platelet-derived growth factor-B (PDGF-B) expression in B16-F10 cells and VEGF receptor expression in HMEC-1 endothelial cells. When the antitumor effect of TAP7f was studied in C57BL/6J mice challenged with B16-F10 melanoma cells, a significant reduction of tumor growth was observed. Furthermore, a decreased expression of VEGF, PDGF-B, and the endothelial cell marker CD34 was observed in tumors from TAP7f-treated mice. Together, our results suggest that the antiangiogenic activity of TAP7f contributes to its antitumor and antimetastatic action and positions this penicillin derivative as an alternative or complementary agent for the treatment of melanoma. KEY MESSAGES: • TAP7f inhibits proliferation, migration, tube formation, and actin cytoskeleton organization of endothelial cells. • TAP7f downregulates VEGF receptor expression in endothelial cells. • TAP7f downregulates VEGF and PDGF expression in melanoma cells. • TAP7f inhibits angiogenesis in vivo.

Characterization of pulmonary vascular remodeling and MicroRNA-126-targets in COPD-pulmonary hypertension

BackgroundDespite causing increased morbidity and mortality, pulmonary hypertension (PH) in chronic obstructive pulmonary disease (COPD) patients (COPD-PH) lacks treatment, due to incomplete understanding of its pathogenesis. Hypertrophy of pulmonary arterial walls and pruning of the microvasculature with loss of capillary beds are known features of pulmonary vascular remodeling in COPD. The remodeling features of pulmonary medium- and smaller vessels in COPD-PH lungs are less well described and may be linked to maladaptation of endothelial cells to chronic cigarette smoking (CS). MicroRNA-126 (miR126), a master regulator of endothelial cell fate, has divergent functions that are vessel-size specific, supporting the survival of large vessel endothelial cells and inhibiting the proliferation of microvascular endothelial cells. Since CS decreases miR126 in microvascular lung endothelial cells, we set out to characterize the remodeling by pulmonary vascular size in COPD-PH and its relationship with miR126 in COPD and COPD-PH lungs.MethodsDeidentified lung tissue was obtained from individuals with COPD with and without PH and from non-diseased non-smokers and smokers. Pulmonary artery remodeling was assessed by ⍺-smooth muscle actin (SMA) abundance via immunohistochemistry and analyzed by pulmonary artery size. miR126 and miR126-target abundance were quantified by qPCR. The expression levels of ceramide, ADAM9, and endothelial cell marker CD31 were assessed by immunofluorescence.ResultsPulmonary arteries from COPD and COPD-PH lungs had significantly increased SMA abundance compared to non-COPD lungs, especially in small pulmonary arteries and the lung microvasculature. This was accompanied by significantly fewer endothelial cell markers and increased pro-apoptotic ceramide abundance. miR126 expression was significantly decreased in lungs of COPD individuals. Of the targets tested (SPRED1, VEGF, LAT1, ADAM9), lung miR126 most significantly inversely correlated with ADAM9 expression. Compared to controls, ADAM9 was significantly increased in COPD and COPD-PH lungs, predominantly in small pulmonary arteries and lung microvasculature.ConclusionBoth COPD and COPD-PH lungs exhibited significant remodeling of the pulmonary vascular bed of small and microvascular size, suggesting these changes may occur before or independent of the clinical development of PH. Decreased miR126 expression with reciprocal increase in ADAM9 may regulate endothelial cell survival and vascular remodeling in small pulmonary arteries and lung microvasculature in COPD and COPD-PH.

Glial Response to Intranasal Mesenchymal Stem Cells in Intermittent Cuprizone Model of Demyelination.

Intranasal mesenchymal stem cells (MSCs) delivery is a non-invasive method that has received interests for treatment of neurodegenerative diseases, such as multiple sclerosis (MS). The impact of intranasal MSCs on intermittent cuprizone model of demyelination was a focus of this study. C57/BL6 mice were fed with 0.3% cuprizone in an intermittent or single ways. Luxol fast blue (LFB), Rotarod test, quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry and western blot (WB) were used for interpretation of outcomes. MSCs effectively homed to the corpus callosum area, were able to improve motor coordination and to promote myelin recovery in the intermittent cuprizone (INTRCPZ/MSCs). Astrogliosis (GFAP+ cells) and microgliosis (Iba-1+ cells) were hampered, and more mature oligodendrocyte cells (APC+ cells) were identified in mice receiving INTRCPZ/MSCs. Such treatment also considerably reduced markers related to the macrophage type 1 (M1) cells, namely iNOS and CD86, but it recovered the M2 markers MRC-1 and TREM-2. In addition, a remarkable decrease in the expressions of pro-inflammatory IL-1β and TNFα but an increase in the rate of anti-inflammatory TGF-β and IL-10 were identified in mice that underwent INTRCPZ/MSCs therapy. Finally, microvascular changes were evaluated, and a noticeable increase in the expression of the endothelial cell marker CD31 was found in the INTRCPZ/MSCs-treated mice (p < 0.05 for all). The outcomes are representative of the efficacy of intranasal MSCs delivery in intermittent cuprizone model of MS for reshaping macrophage polarity along with modification of glial, inflammatory, and angiogenic markers in favor of therapy.

TNFAIP3 mediates FGFR1 activation-induced breast cancer angiogenesis by promoting VEGFA expression and secretion.

To investigate the role and mechanism of TNF-inducible protein 3(TNFAIP3) in breast cancer angiogenesis induced by fibroblast growth factor receptor1 (FGFR1) activation. The immunohistochemical assay was used to detect the expression of vascular endothelial cell marker CD31 and CD105 in mice DCIS.COM-iFGFR1 transplanted tumor (previously established by our group). The effects of TNFAIP3 knockout/knockdown breast cancer cell lines on angiogenesis, migration, and invasion of Human Umbilical Vein Endothelial Cells (HUVEC) were detected by the tubulogenesis and Trewells assay. RNA-seq analysis of TNFAIP3 downstreams differential genes after TNFAIP3 knockdown. The expression and secretion of VEGFA after FGFR1 activation in breast cancer cells were detected by qPCR, Western blot, and ELISA. Immunohistochemistry showed that TNFAIP3 knockout inhibited the expression of CD31 and CD105 in DCIS grafted tumors promoted by FGFR1 activation. Tubulogenesis and Trewells experiments showed that TNFAIP3 gene knockout/knockdown inhibited the angiogenesis, migration, and invasion of HUVEC cells promoted by FGFR1 activation. qPCR assay showed that VEGFA mRNA level in the TNFAIP3 knockdown cell line was significantly down-regulated (p < 0.05). qPCR, Western blot and ELISA results showed that TNFAIP3 gene knockout/knockdown could inhibit the expression and secretion of VEGFA in breast cancer cells induced by FGFR1 activation. TNFAIP3 promotes breast cancer angiogenesis induced by FGFR1 activation through the expression and secretion of VEGFA.

Vascular endothelial growth factor-165b protects the blood-retinal barrier from damage after acute high intraocular pressure in rats.

To elucidate the role of vascular endothelial growth factor-165b (VEGF-165b) in blood-retinal barrier (BRB) injury in the rat acute glaucoma model. In this study, the rat acute high intraocular pressure (HIOP) model was established before and after intravitreous injection of anti-VEGF-165b antibody. The expression of VEGF-165b and zonula occludens-1 (ZO-1) in rat retina was detected by double immunofluorescence staining and Western blotting, and the breakdown of BRB was detected by Evans blue (EB) dye. The intact retina of rats expressed VEGF-165b and ZO-1 protein, which were mainly located in the retinal ganglion cell layer and the inner nuclear layer and were both co-expressed with vascular endothelial cell markers CD31. After acute HIOP, the expression of VEGF-165b was up-regulated; the expression of ZO-1 was down-regulated at 12h and then recovered at 3d; EB leakage increased, peaking at 12h. After intravitreous injection of anti-VEGF-165b antibody, the expression of VEGF-165b protein was no significantly changed; and the down-regulation of the expression of ZO-1 was more obvious; EB leakage became more serious, peaking at 3d. EB analysis also showed that EB leakage in the peripheral retina was greater than that in the central retina. The endogenous VEGF-165b protein may protect the BRB from acute HIOP by regulating the expression of ZO-1. The differential destruction of BRB after acute HIOP may be related to the selective loss of retinal ganglion cells.

Exosomes Derived from Secretome Human Umbilical Vein Endothelial Cells (Exo-HUVEC) Ameliorate the Photo-Aging of Skin Fibroblast.

PurposeThis is an in-vitro experimental study to analyze the effect of Exo-HUVEC on endothelial cell (CD31), cell proliferation, matrix metalloproteinase 1 (MMP-1) and collagen type 1 on irradiated fibroblast with UVB as photo-aging model.Patients and MethodsFibroblast cultures were divided into 5 groups, namely without UVB exposure, UVB exposure 600mJ/cm2 for 80 seconds as photo-aging model, and UVB exposure +Exo-HUVEC exposure 0.1%, 0.5% and 1%. The endothelial cell was stained with a CD31 marker, MMP-1 were examined with ELISA, cell proliferation is detected using an MTT assay; meanwhile, collagen type 1 deposition and endothelial cell were measured using flowcytometry.ResultsThis study found positive endothelial cell marker CD31. Significant difference was found in cell proliferation, MMP-1 and collagen type 1 level between the control group with UVB irradiation and the treatment group with Exo-HUVEC (p < 0.05).ConclusionExo-HUVEC significantly increases cell proliferation and collagen type 1 level, while decrease MMP-1 levels on irradiated fibroblast; therefore, Exo-HUVEC ameliorate the photo-aging of skin fibroblast.