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.

Exosomes derived from BMSCs in osteogenic differentiation promote type H blood vessel angiogenesis through miR-150-5p mediated metabolic reprogramming of endothelial cells.

Osteogenesis is tightly coupled with angiogenesis spatiotemporally. Previous studies have demonstrated that type H blood vessel formed by endothelial cells with high expression of CD31 and Emcn (CD31hi Emcnhi ECs) play a crucial role in bone regeneration. The mechanism of the molecular communication around CD31hi Emcnhi ECs and bone mesenchymal stem cells (BMSCs) in the osteogenic microenvironment is unclear. This study indicates that exosomes from bone mesenchymal stem cells with 7 days osteogenic differentiation (7D-BMSCs-exo) may promote CD31hi Emcnhi ECs angiogenesis, which was verified by tube formation assay, qRT-PCR, Western blot, immunofluorescence staining and µCT assays etc. in vitro and in vivo. Furthermore, by exosomal miRNA microarray and WGCNA assays, we identified downregulated miR-150-5p as the most relative hub gene coupling osteogenic differentiation and type H blood vessel angiogenesis. With bioinformatics assays, dual luciferase reporter experiments, qRT-PCR and Western blot assays, SOX2(SRY-Box Transcription Factor 2) was confirmed as a novel downstream target gene of miR-150-5p in exosomes, which might be a pivotal mechanism regulating CD31hi Emcnhi ECs formation. Additionally, JC-1 immunofluorescence staining, Western blot and seahorse assay results showed that the overexpression of SOX2 could shift metabolic reprogramming from oxidative phosphorylation (OXPHOS) to glycolysis to enhance the CD31hi Emcnhi ECs formation. The PI3k/Akt signaling pathway might play a key role in this process. In summary, BMSCs in osteogenic differentiation might secrete exosomes with low miR-150-5p expression to induce type H blood vessel formation by mediating SOX2 overexpression in ECs. These findings might reveal a molecular mechanism of osteogenesis coupled with type H blood vessel angiogenesis in the osteogenic microenvironment and provide a new therapeutic target or cell-free remedy for osteogenesis impaired diseases.

Plasminogen activator inhibitor-1 is involved in glucocorticoid-induced decreases in angiogenesis during bone repair in mice.

INTRODUCTION: Glucocorticoids delay fracture healing and induce osteoporosis. Angiogenesis plays an important role in bone repair after bone injury. Plasminogen activator inhibitor-1 (PAI-1) is the principal inhibitor of plasminogen activators and an adipocytokine that regulates metabolism. However, the mechanisms by which glucocorticoids delay bone repair remain unclear. MATERIALS AND METHODS: Therefore, we herein investigated the roles of PAI-1 and angiogenesis in glucocorticoid-induced delays in bone repair after femoral bone injury using PAI-1-deficient female mice intraperitoneally administered dexamethasone (Dex). RESULTS: PAI-1 deficiency significantly attenuated Dex-induced decreases in the number of CD31-positive vessels at damaged sites 4 days after femoral bone injury in mice. PAI-1 deficiency also significantly ameliorated Dex-induced decreases in the number of CD31- and endomucin-positive type H vessels and CD31-positive- and endomucin-negative vessels at damaged sites 4 days after femoral bone injury. Moreover, PAI-1 deficiency significantly mitigated Dex-induced decreases in the expression of vascular endothelial growth factor as well as hypoxia inducible factor-1α, transforming growth factor-ß1, and bone morphogenetic protein-2 at damaged sites 4 days after femoral bone injury. CONCLUSION: The present results demonstrate that Dex-reduced angiogenesis at damaged sites during the early bone-repair phase after femoral bone injury partly through PAI-1 in mice.

Numerous multinucleated giant cells in cutaneous epithelioid angiosarcoma and pulmonary metastasis: A unique observation with potential diagnostic pitfalls.

The histopathologic diagnosis of poorly differentiated cutaneous angiosarcoma can be challenging. We report a case of cutaneous epithelioid angiosarcoma with numerous multinucleated giant cells (MGCs) developing pulmonary metastasis. A 79-year-old man presented with a red-purple plaque on the scalp. A skin biopsy revealed epithelioid cell proliferation, admixed with numerous MGCs, and background hemorrhage. Vascular spaces were focally present and lined by atypical endothelial cells, including MGCs. Immunohistochemically, tumor cells, including MGCs, were positive for CD31, D2-40, and ERG. The patient received radiation therapy and chemotherapy, after which a follow-up CT scan revealed symptomless pneumothorax and pulmonary metastases. The patient received palliative partial lung resection, and the specimen revealed histopathological and immunohistochemical features similar to the primary cutaneous lesion. Our report expands the morphologic spectrum of cutaneous epithelioid angiosarcoma. Cutaneous angiosarcoma is an aggressive neoplasm; thus, awareness of this rare manifestation is important.

Endothelium-Derived Extracellular Vesicles Expressing Intercellular Adhesion Molecules Reflect Endothelial Permeability and Sepsis Severity.

BACKGROUND: Currently, clinical indicators for evaluating endothelial permeability in sepsis are unavailable. Endothelium-derived extracellular vesicles (EDEVs) are emerging as biomarkers of endothelial injury. Platelet endothelial cell adhesion molecule (PECAM) and vascular endothelial (VE)-cadherin are constitutively expressed endothelial intercellular adhesion molecules that regulate intercellular adhesion and permeability. Herein, we investigated the possible association between EDEVs expressing intercellular adhesion molecules (PECAM+ or VE-cadherin+ EDEVs) and endothelial permeability and sepsis severity. METHODS: Human umbilical vein endothelial cells (HUVECs) were stimulated with tumor necrosis factor alpha (TNF-α) directly or after pretreatment with permeability-modifying reagents such as angiopoietin-1, prostacyclin, or vascular endothelial growth factor (VEGF) to alter TNF-α-induced endothelial hyperpermeability. Endothelial permeability was measured using the dextran assay or transendothelial electrical resistance. Additionally, a prospective cross-sectional observational study was conducted to analyze circulating EDEV levels in patients with sepsis. EDEVs were examined in HUVEC culture supernatants or patient plasma (nonsepsis, n = 30; sepsis, n = 30; septic shock, n = 42) using flow cytometry. The Wilcoxon rank-sum test was used for comparisons between 2 groups. Comparisons among 3 or more groups were performed using the Steel-Dwass test. Spearman's test was used for correlation analysis. Statistical significance was set at P < .05. RESULTS: TNF-α stimulation of HUVECs significantly increased EDEV release and endothelial permeability. Pretreatment with angiopoietin-1 or prostacyclin suppressed the TNF-α-induced increase in endothelial permeability and inhibited the release of PECAM+ and VE-cadherin+ EDEVs. In contrast, pretreatment with VEGF increased TNF-α-induced endothelial permeability and the release of PECAM+ and VE-cadherin+ EDEVs. However, pretreatment with permeability-modifying reagents did not affect the release of EDEVs expressing inflammatory stimulus-inducible endothelial adhesion molecules such as E-selectin, intracellular adhesion molecule-1, or vascular cell adhesion molecule-1. The number of PECAM+ EDEVs on admission in the septic-shock group (232 [124, 590]/µL) was significantly higher (P = .043) than that in the sepsis group (138 [77,267]/µL), with an average treatment effect of 98/µL (95% confidence interval [CI], 2-270/µL), and the number of VE-cadherin+ EDEVs in the septic-shock group (173 [76,339]/µL) was also significantly higher (P = .004) than that in the sepsis group (81 [42,159]/µL), with an average treatment effect (ATE) of 79/µL (95% CI, 19-171/µL); these EDEV levels remained elevated until day 5. CONCLUSIONS: EDEVs expressing intercellular adhesion molecules (PECAM+ or VE-cadherin+ EDEVs) may reflect increased endothelial permeability and could be valuable diagnostic and prognostic markers for sepsis.

Ghrelin induced by ultraviolet B exposure promotes the restoration of diabetic cutaneous wound healing.

BACKGROUND: Diabetes mellitus (DM) presents impediment to wound healing. While ultraviolet B (UVB) exposure showed therapeutic potential in various skin conditions, its capacity to mediate diabetic wound healing remains unclear. To investigate the efficacy of UVB on wound healing and its underlying basis. MATERIALS AND METHODS: Male C57BL/6 mice were subjected to the high-fat diet followed by streptozotocin administration to establish the diabetic model. Upon confirmation of diabetes, full-thickness wounds were inflicted and the treatment group received UVB radiation at 50 mJ/cm2 for 5 min every alternate day for 2 weeks. Wound healing rate was then assessed, accompanied by evaluations of blood glucose, lipid profiles, CD31 expression, and concentrations of ghrelin and leptin. Concurrently, in vitro studies were executed to evaluate the protective role of ghrelin on human umbilical vein endothelial cells (HUVEC) under high glucose (HG) conditions. RESULTS: Post UVB exposure, there was a marked acceleration in wound healing in DM mice without alterations in hyperglycemia and lipid profiles. Compared to non-UVB-exposed mice, the UVB group showed enhanced angiogenesis manifested by a surge in CD31 expression. This trend appeared to be in harmony with the elevated ghrelin levels. In vitro experiments indicated that ghrelin significantly enhanced the migratory pace and angiogenic properties of HUVEC under HG-induced stress, potentially mediated by an upregulation in vascular endothelial growth factor expression. CONCLUSION: UVB exposure bolstered wound healing in diabetic mice, plausibly mediated through augmented angiogenesis induced by ghrelin secretion. Such findings underscore the vast potential of UVB-induced ghrelin in therapeutic strategies targeting diabetic wound healing.

In the Rat Hippocampus, Pilocarpine-Induced Status Epilepticus Is Associated with Reactive Glia and Concomitant Increased Expression of CD31, PDGFRß, and Collagen IV in Endothelial Cells and Pericytes of the Blood-Brain Barrier.

In humans and animal models, temporal lobe epilepsy (TLE) is associated with reorganization of hippocampal neuronal networks, gliosis, neuroinflammation, and loss of integrity of the blood-brain barrier (BBB). More than 30% of epilepsies remain intractable, and characterization of the molecular mechanisms involved in BBB dysfunction is essential to the identification of new therapeutic strategies. In this work, we induced status epilepticus in rats through injection of the proconvulsant drug pilocarpine, which leads to TLE. Using RT-qPCR, double immunohistochemistry, and confocal imaging, we studied the regulation of reactive glia and vascular markers at different time points of epileptogenesis (latent phase-3, 7, and 14 days; chronic phase-1 and 3 months). In the hippocampus, increased expression of mRNA encoding the glial proteins GFAP and Iba1 confirmed neuroinflammatory status. We report for the first time the concomitant induction of the specific proteins CD31, PDGFRß, and ColIV-which peak at the same time points as inflammation-in the endothelial cells, pericytes, and basement membrane of the BBB. The altered expression of these proteins occurs early in TLE, during the latent phase, suggesting that they could be associated with the early rupture and pathogenicity of the BBB that will contribute to the chronic phase of epilepsy.

[The influence of knocking down the expression of low-density lipoprotein receptor associated proteins on the vascular abnormalities in hepatocellular carcinoma and its mechanisms].

Objectives: To investigate the effect of the expression of low-density lipoprotein receptor associated protein (LDLR) on the vascular abnormalities in hepatocellular carcinoma (HCC) and its mechanisms. Methods: Based on the information of Oncomine Cancer GeneChip database, we analyzed the correlation between the expression level of LDLR and the expression level of carcinoembryonic antigen (CEA) and CD31 in hepatocellular carcinoma tissues. Lentiviral transfection of short hairpin RNA target genes was used to construct LDLR-knockdown MHCC-97H and HLE hepatocellular carcinoma cells. The differential genes and their expression level changes in LDLR-knockdown hepatocellular carcinoma cells were detected by transcriptome sequencing, real-time fluorescence quantitative polymerase chain reaction, and protein immunoblotting. The gene-related signaling pathways that involve LDLR were clarified by enrichment analysis. The effect of LDLR on CEA was assessed by the detection of CEA content in conditioned medium of hepatocellular carcinoma cells. Angiogenesis assay was used to detect the effect of LDLR on the angiogenic capacity of human umbilical vein endothelial cells, as well as the role of CEA in the regulation of angiogenesis by LDLR. Immunohistochemical staining was used to detect the expression levels of LDLR in 176 hepatocellular carcinoma tissues, and CEA and CD31 in 146 hepatocellular carcinoma tissues, and analyze the correlations between the expression levels of LDLR, CEA, and CD31 in the tissues, serum CEA, and alanine transaminase (ALT). Results: Oncomine database analysis showed that the expressions of LDLR and CEA in the tissues of hepatocellular carcinoma patients with portal vein metastasis were negatively correlated (r=-0.64, P=0.001), whereas the expressions of CEA and CD31 in these tissues were positively correlated ( r=0.46, P=0.010). The transcriptome sequencing results showed that there were a total of 1 032 differentially expressed genes in the LDLR-knockdown group and the control group of MHCC-97H cells, of which 517 genes were up-regulated and 515 genes were down-regulated. The transcript expression level of CEACAM5 was significantly up-regulated in the cells of the LDLR-knockdown group. The Gene Ontology (GO) function enrichment analysis showed that the differential genes were most obviously enriched in the angiogenesis function. The Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis showed that the relevant pathways involved mainly included the cellular adhesion patch, the extracellular matrix receptor interactions, and the interactions with the extracellular matrix receptors. The CEA content in the conditioned medium of the LDLR-knockdown group was 43.75±8.43, which was higher than that of the control group (1.15±0.14, P＜0.001). The results of angiogenesis experiments showed that at 5 h, the number of main junctions, the number of main segments, and the total area of the lattice formed by HUVEC cells cultured with the conditioned medium of MHCC-97H cells in the LDLR-knockdown group were 295.3±26.4, 552.5±63.8, and 2 239 781.0±13 8211.9 square pixels, which were higher than those of the control group (113.3±23.5, 194.8±36.5, and 660 621.0±280 328.3 square pixels, respectively, all P＜0.01).The number of vascular major junctions, the number of major segments, and the total area of the lattice formed by HUVEC cells cultured in conditioned medium with HLE cells in the LDLR-knockdown group were 245.3±42.4, 257.5±20.4, and 2 535 754.5±249 094.2 square pixels, respectively, which were all higher than those of the control group (113.3±23.5, 114.3±12.2, and 1 565 456.5±219 259.7 square pixels, respectively, all P＜0.01). In the conditioned medium for the control group of MHCC-97H cells,the number of main junctions, the number of main segments, and the total area of the lattice formed by the addition of CEA to cultured HUVEC cells were 178.9±12.0, 286.9±12.3, and 1 966 990.0±126 249.5 spixels, which were higher than those in the control group (119.7±22.1, 202.7±33.7, and 1 421 191.0±189 837.8 square pixels, respectively). The expression of LDLR in hepatocellular carcinoma tissues was not correlated with the expression of CEA, but was negatively correlated with the expression of CD31 (r=-0.167, P=0.044), the level of serum CEA (r=-0.061, P=0.032), and the level of serum ALT(r=-0.147,P=0.05). The expression of CEA in hepatocellular carcinoma tissues was positively correlated with the expression of CD31 (r=0.192, P=0.020). The level of serum CEA was positively correlated with the level of serum ALT (r=0.164, P=0.029). Conclusion: Knocking down LDLR can promote vascular abnormalities in HCC by releasing CEA.

Pathological Changes in the Gastrocnemius Muscle throughout Hind Limb Ischemia Modeling in Rats.

We present a two-stage model for the study of chronic hind limb ischemia in rats. In the area of ischemia, sclerotic changes with atrophic rhabdomyocytes and reduced vascularization were revealed. CD31 expression in the endothelium increased proportionally to the number of vessels in the ischemic zone, and at the same time, focal expression of ßIII-tubulin was detected in the newly formed nerve fibers. These histological features are equivalent to the development of peripheral arterial disease in humans, which allows using our model in the search for new therapeutic strategies.

TGFß1 Regulates Cellular Composition of In Vitro Cardiac Perivascular Niche Based on Cardiospheres.

The cardiac perivascular niche is a cellular microenvironment of a blood vessel. The principles of niche regulation are still poorly understood. We studied the effect of TGFß1 on cells forming the cardiac perivascular niche using 3D cell culture (cardiospheres). Cardiospheres contained progenitor (c-Kit), endothelial (CD31), and mural (αSMA) cells, basement membrane proteins (laminin) and extracellular matrix proteins (collagen I, fibronectin). TGFß1 treatment decreased the length of CD31+ microvasculature, VE cadherin protein level, and proportion of NG2+ cells, and increased proportion of αSMA+ cells and transgelin/SM22α protein level. We supposed that this effect is related to the stabilizing function of TGFß1 on vascular cells: decreased endothelial cell proliferation, as shown for HUVEC, and activation of mural cell differentiation.

[Analysis of intracranial saccular aneurysm wall: neuroimaging and histopathological correlates]. / Issledovanie stenki intrakranial'nykh meshotchatykh anevrizm: neirovizualizatsionnye i gistopatologicheskie korrelyaty.

BACKGROUND: Contrast enhancement of intracranial aneurysm wall during MRI with targeted visualization of vascular wall correlates with previous aneurysm rupture and, according to some data, may be a predictor of further rupture of unruptured aneurysms. OBJECTIVE: To analyze possible causes of aneurysm contrast enhancement considering morphological data of aneurysm walls. MATERIAL AND METHODS: The study included 44 patients with intracranial aneurysms who underwent preoperative MRI between November 2020 and September 2022. Each aneurysm was assessed regarding contrast enhancement pattern. Microsurgical treatment of aneurysm was accompanied by resection of its wall for subsequent histological and immunohistochemical analysis regarding thrombosis, inflammation and neovascularization. Specimens were subjected to histological and immunochemical analysis. Immunohistochemical analysis was valuable to estimate inflammatory markers CD68 and CD3, as well as neurovascularization marker SD31. RESULTS: Aneurysms with contrast-enhanced walls were characterized by higher number of CD3+, CD68+, CD31+ cells and parietal clots. Intensity of contrast enhancement correlated with aneurysm wall abnormalities. CONCLUSION: Contrast enhancement of aneurysm wall can characterize various morphological abnormalities.

[Protective effect of FAK inhibitor PF-562271 against human umbilical vein endothelial cell injury induced by aging platelets].

OBJECTIVE: To investigate the protective effect of PF-562271, a FAK inhibitor, against aging platelet-induced injury in human umbilical vein endothelial cells (HUVECs). METHODS: Cultured HUVECs were treated with vehicle, lipopolysaccharide (LPS), LPS+aging platelets, or LPS+aging platelets+PF-562271. The changes in protein expressions of FAK, pFAK and PECAM-1 in the treated cells were detected using Western blotting and immunofluorescence assay, and the level of reactive oxygen species (ROS) was detected with flow cytometry. The changes of barrier function of the cells were assessed with cell permeability test and transendothelial cell resistance test. RT-qPCR was used to analyze mRNA expressions of inflammatory factors, and pro-inflammatory cytokine levels in the culture supernatants was determined with enzyme-linked immunosorbent assay. Immunofluorescence assay was used to examine the effect of the ROS inhibitor vitamin C on PECAM-1 expression in the cells with different treatments. RESULTS: Treatment of HUVECs with LPS and aging platelets significantly increased cellular protein expressions of FAK, pFAK and PECAM-1, which were effectively lowered by addition of PF-562271 (P < 0.05). LPS and aged platelets obviously enhanced ROS production in the cells, which was inhibited by the addition of PF-562271 (P < 0.001). PF-562271 significantly alleviated the damage of endothelial cell barrier function of the cells caused by LPS and aging platelets (P < 0.01). The expressions of TNF-α, IL-6 and IL-8 in HUVECs increased significantly after exposure to LPS and aging platelets, and were obviously lowered after treatment with PF-562271 (P < 0.05). Treatment with vitamin C significantly decreased the expression of PECAM-1 protein in the cells (P < 0.01). CONCLUSION: The FAK inhibitor PF-562271 alleviates endothelial cell damage induced by LPS and aging platelets by lowering cellular oxidative stress levels and reducing inflammatory responses.

Transient heat stress protects from severe endothelial damage and dysfunction during prolonged experimental ex-vivo lung perfusion.

Introduction: The pulmonary endothelium is the primary target of lung ischemia-reperfusion injury leading to primary graft dysfunction after lung transplantation. We hypothesized that treating damaged rat lungs by a transient heat stress during ex-vivo lung perfusion (EVLP) to elicit a pulmonary heat shock response could protect the endothelium from severe reperfusion injury. Methods: Rat lungs damaged by 1h warm ischemia were reperfused on an EVLP platform for up to 6h at a constant temperature (T°) of 37°C (EVLP37°C group), or following a transient heat stress (HS) at 41.5°C from 1 to 1.5h of EVLP (EVLPHS group). A group of lungs exposed to 1h EVLP only (pre-heating conditions) was added as control (Baseline group). In a first protocol, we measured lung heat sock protein expression (HSP70, HSP27 and Hsc70) at selected time-points (n=5/group at each time). In a second protocol, we determined (n=5/group) lung weight gain (edema), pulmonary compliance, oxygenation capacity, pulmonary artery pressure (PAP) and vascular resistance (PVR), the expression of PECAM-1 (CD31) and phosphorylation status of Src-kinase and VE-cadherin in lung tissue, as well as the release in perfusate of cytokines (TNFα, IL-1ß) and endothelial biomarkers (sPECAM, von Willebrand Factor -vWF-, sE-selectin and sICAM-1). Histological and immunofluorescent studies assessed perivascular edema and formation of 3-nitrotyrosine (a marker of peroxinitrite) in CD31 lung endothelium. Results: HS induced an early (3h) and persisting expression of HSP70 and HSP27, without influencing Hsc70. Lungs from the EVLP37°C group developed massive edema, low compliance and oxygenation, elevated PAP and PVR, substantial release of TNFα, IL-1ß, s-PECAM, vWF, E-selectin and s-ICAM, as well as significant Src-kinase activation, VE-cadherin phosphorylation, endothelial 3-NT formation and reduced CD31 expression. In marked contrast, all these alterations were either abrogated or significantly attenuated by HS treatment. Conclusion: The therapeutic application of a transient heat stress during EVLP of damaged rat lungs reduces endothelial permeability, attenuates pulmonary vasoconstriction, prevents src-kinase activation and VE-cadherin phosphorylation, while reducing endothelial peroxinitrite generation and the release of cytokines and endothelial biomarkers. Collectively, these data demonstrate that therapeutic heat stress may represent a promising strategy to protect the lung endothelium from severe reperfusion injury.

Effect of electroacupuncture intervention on angiogenesis in psoriasis mice. / ç”µé’ˆå¯¹é“¶å±‘ç— å°é¼ è¡€ç®¡ç”Ÿæˆçš„å½±å“åŠæœºåˆ¶ç ”ç©¶.

OBJECTIVES: To observe the effect of electroacupuncture (EA) stimulation of "Zusanli"(ST36) and"Xuehai"(SP10) on the angiogenesis of the local injured skin tissue in mice with psoriasis, so as to explore its mechanisms underlying improvement of psoriasis-induced skin lesions. METHODS: A total of 24 female BALB/c mice aged 6-8 weeks were randomly divided into control, model and EA groups, with 8 mice in each group. The psoriasis-like skin lesion model was established by application of 5% imiquimod (IMQ) cream to the mice's back skin, 62.5 mg/d, for 7 days after local depilation, and the mice of the control group received local application of an equal amount of petroleum jelly once a day for 7 days. EA stimulation (2 Hz/100 Hz) was applied to ST36 and SP10 for 30 min, once daily for 7 consecutive days. Photos of the topical injured skin at the back were taken every day, and the severity of psoriasis lesions (psoriasis area and severity index ï¼»PASIï¼½) was scaled. Following H.E. staining, the morphological changes in the injured skin tissue were observed with epidermal thickness analyzed, and the Masson staining was used to observe the proportion of collagen fibers in the injured skin tissues. Immunohistochemical method was used to detect the expression of microvascular markers CD31 and vascular endothelial growth factor (VEGF) and the microvascular density (MVD) was calculated. Western blot was used to detect the expression levels of CD31, VEGF proteins and mitogen activated protein kinases (MAPK) signaling pathway related proteins p38, phosphorylated p38 (p-p38), extracellular regulated protein kinases (ERK), p-ERK, c-Jun N-terminal kinase (JNK) and p-JNK in the injured skin tissue. RESULTS: Compared with the control group, the mice in the model group showed an evident increase in the erythema score, scales score, skin thickening score and PASI score, epidermal thickness, proportion of the collagen fibers, MVD value of CD31 and VEGF, and expression levels of CD31 and VEGF proteins, and p-p38/p38, p-ERK/ERK and p-JNK/JNK ratios in the injured skin tissue (P<0.001, P<0.01). In contrast to the model group, the EA group had a significant decrease in the levels of all the indexes mentioned above (P<0.05, P<0.01, P<0.001). CONCLUSIONS: EA intervention can improve the psoriasis-like skin lesions induced by IMQ in mice, which may be related with its functions in down-regulating the expression of angiogenic related factors CD31 and VEGF proteins and MAPK signaling pathway related proteins in the topical injured skin tissue.

Prostate Specific Membrane Antigen Expression in a Syngeneic Breast Cancer Mouse Model.

PURPOSE: Prostate specific membrane antigen (PSMA) has been studied in human breast cancer (BCa) biopsies, however, lack of data on PSMA expression in mouse models impedes development of PSMA-targeted therapies, particularly in improving breast conserving surgery (BCS) margins. This study aimed to validate and characterize the expression of PSMA in murine BCa models, demonstrating that PSMA can be utilized to improve therapies and imaging techniques. METHODS: Murine triple negative breast cancer 4T1 cells, and human cell lines, MDA-MB-231, MDA-MB-468, implanted into the mammary fat pads of BALB/c mice, were imaged by our PSMA targeted theranostic agent, PSMA-1-Pc413, and tumor to background ratios (TBR) were calculated to validate selective uptake. Immunohistochemistry was used to correlate PSMA expression in relation to CD31, an endothelial cell biomarker highlighting neovasculature. PSMA expression was also quantified by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). RESULTS: Accumulation of PSMA-1-Pc413 was observed in 4T1 primary tumors and associated metastases. Average TBR of 4T1 tumors were calculated to be greater than 1.5-ratio at which tumor tissues can be distinguished from normal structures-at peak accumulation with the signal intensity in 4T1 tumors comparable to that in high PSMA expressing PC3-pip tumors. Extraction of 4T1 tumors and lung metastases followed by RT-PCR analysis and PSMA-CD31 co-staining shows that PSMA is consistently localized on tumor neovasculature with no expression in tumor cells and surrounding normal tissues. CONCLUSION: The selective uptake of PSMA-1-Pc413 in these cancer tissues as well as the characterization and validation of PSMA expression on neovasculature in this syngeneic 4T1 model emphasizes their potential for advancements in targeted therapies and imaging techniques for BCa. PSMA holds great promise as an oncogenic target for BCa and its associated metastases.

[A preliminary in vivo and in vitro study of endothelial cell pyroptosis in the periodontal inflammatory environment].

Objective: To observe whether endothelial cells undergo pyroptosis in the inflammatory periodontal environment by using a model in vivo and in vitro, providing an experimental basis for indepth understanding of the underlying pathogenesis of periodontitis. Methods: According to the classification of periodontal diseases of 2018, gingival tissues were collected from periodontally healthy subjects and patients with stage Ⅲ-Ⅳ, grade C periodontitis, who presented Department of Oral and Maxillofacial Surgery and Department of Periodontology, School of Stomatology, The Fourth Military Medical University from April to May 2022. Immunohistochemical staining was performed to detect the expression level and distribution of gasdermin D (GSDMD), a hallmark protein of cell pyroptosis, in gingival tissues. Periodontitis models were established in each group by ligating the maxillary second molar teeth of three mice for 2 weeks (ligation group). The alveolar bone resorption was determined by micro-CT (mice without ligation treatment were used as the control group), and the colocalization of GSDMD and CD31 were quantitatively analyzed by immunofluorescence staining in gingival tissues of healthy and inflammatory mice. Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and treated with lipopolysaccharide (LPS) of Porphyromonas gingivalis (Pg) combined with adenosine triphosphate (ATP) at various concentrations of 0.5, 1.0, 2.5, 5.0, and 10.0 mg/L, respectively, and the 0 mg/L group was set as the control group at the same time. Scanning electron microscopy was used to observe the morphology of HUVECs. Western blotting was used to detect the expression of gasdermin D-N terminal domains (GSDMD-N) protein and immunofluorescence cell staining was used to detect the expression and distribution of GSDMD. Cell counting kit-8 (CCK-8) was used to detect the proliferative ability of HUVECs, and propidium iodide (PI) staining was used to detect the integrity of cell membrane of HUVECs. Results: Immunohistochemistry showed that GSDMD in gingival tissues of periodontitis was mainly distributed around blood vessels and its expression level was higher than that in healthy tissues. Micro-CT showed that alveolar bone resorption around the maxillary second molar significantly increased in ligation group mice compared with control subjects (t=8.88, P<0.001). Immunofluorescence staining showed significant colocalization of GSDMD with CD31 in the gingival vascular endothelial cells in mice of ligation group. The results of scanning electron microscopy showed that there were pores of different sizes, the typical morphology of pyroptosis, on HUVECs cell membranes in the inflammatory environment simulated by ATP combined with different concentrations of LPS, and 2.5 mg/L group showed the most dilated and fused pores on cell membranes, with the cells tended to lyse and die. Western blotting showed that the expression of GSDMD-N, the hallmark protein of cell pyroptosis, was significantly higher in 2.5 and 5.0 mg/L groups than that in the control group (F=3.86, P<0.01). Immunofluorescence cell staining showed that the average fluorescence intensity of GSDMD in 2.5 mg/L group elevated the most significantly in comparison with that in the control group (F=35.25, P<0.001). The CCK-8 proliferation assay showed that compared to the control group (1.00±0.02), 0.5 mg/L (0.52±0.07), 1.0 mg/L (0.57±0.10), 2.5 mg/L (0.58±0.04), 5.0 mg/L (0.55±0.04), 10.0 mg/L (0.61±0.03) groups inhibited cell proliferation (F=39.95, P<0.001). PI staining showed that the proportion of positive stained cells was highest [(56.07±3.22)%] in 2.5 mg/L group (F=88.24, P<0.001). Conclusions: Endothelial cells undergo significant pyroptosis in both in vivo and in vitro periodontal inflammatory environments, suggesting that endothelial cell pyroptosis may be an important pathogenic factor contributing to the pathogenesis of periodontitis.

Tongxinluo Activates PI3K/AKT Signaling Pathway to Inhibit Endothelial Mesenchymal Transition and Attenuate Myocardial Fibrosis after Ischemia-Reperfusion in Mice.

OBJECTIVE: To investigate the potential role of Tongxinluo (TXL) in attenuating myocardial fibrosis after myocardial ischemia-reperfusion injury (MIRI) in mice. METHODS: A MIRI mouse model was established by left anterior descending coronary artery ligation for 45 min. According to a random number table, 66 mice were randomly divided into 6 groups (n=11 per group): the sham group, the model group, the LY-294002 group, the TXL group, the TXL+LY-294002 group and the benazepril (BNPL) group. The day after modeling, TXL and BNPL were administered by gavage. Intraperitoneal injection of LY-294002 was performed twice a week for 4 consecutive weeks. Echocardiography was used to measure cardiac function in mice. Masson staining was used to evaluate the degree of myocardial fibrosis in mice. Qualitative and quantitative analysis of endothelial mesenchymal transition (EndMT) after MIRI was performed by immunohistochemistry, immunofluorescence staining and flow cytometry, respectively. The protein expressions of platelet endothelial cell adhesion molecule-1 (CD31), α-smoth muscle actin (α-SMA), phosphatidylinositol-3-kinase (PI3K) and phospho protein kinase B (p-AKT) were assessed using Western blot. RESULTS: TXL improved cardiac function in MIRI mice, reduced the degree of myocardial fibrosis, increased the expression of CD31 and inhibited the expression of α-SMA, thus inhibited the occurrence of EndMT (P<0.05 or P<0.01). TXL significantly increased the protein expressions of PI3K and p-AKT (P<0.05 or P<0.01). There was no significant difference between TXL and BNPL group (P>0.05). In addition, the use of the PI3K/AKT pathway-specific inhibitor LY-294002 to block this pathway and combination with TXL intervention, eliminated the protective effect of TXL, further supporting the protective effect of TXL. CONCLUSION: TXL activated the PI3K/AKT signaling pathway to inhibit EndMT and attenuated myocardial fibrosis after MIRI in mice.

Endothelial-to-Mesenchymal Transition in Human and Murine Models of Congenital Diaphragmatic Hernia.

INTRODUCTION: Congenital diaphragmatic hernia (CDH) is a complex congenital disorder, characterized by pulmonary hypertension (PH) and hypoplasia. PH secondary to CDH (CDH-PH) features devastating morbidity and mortality (25-30%) among neonates. An unmet need is determining mechanisms triggering CDH-PH to save infants. Prior data suggest abnormal remodeling of the pulmonary vascular extracellular matrix (ECM), presumed to be driven by endothelial-to-mesenchymal transition (EndoMT), hinders postnatal vasodilation and limits anti-PH therapy in CDH. There are limited data on the role of EndoMT in CDH-PH. METHODS: The purpose of the study was to investigate how EndoMT contributes to CDH-PH by identifying cells undergoing EndoMT noted by alpha smooth muscle actin (α-SMA) expression in human umbilical vein endothelial cells (HUVECs) and lung tissue obtained from murine pups using the nitrofen model. N = 8 CDH, N = 8 control HUVECs were stained for α-SMA and CD31 after being exposed for 24 h to TGFB, a known EndoMT promoter. N = 8 nitrofen, N = 8 control murine pup lungs were also stained for α-SMA and CD31. α-SMA and CD31 expression was quantified in HUVECs and murine tissue using Fiji imaging software and normalized to the total number of cells per slide noted by DAPI staining. RESULTS: CDH HUVECs demonstrated a 1.1-fold increase in α-SMA expression (p = 0.02). The murine model did not show statistical significance between nitrofen and control pup lungs; however, there was a 0.4-fold increase in α-SMA expression with a 0.8-fold decrease in CD31 expression in the nitrofen pup lungs when compared to controls. CONCLUSION: These results suggest that EndoMT could potentially play a role in the ECM remodeling seen in CDH-PH.

Diosgenin alleviates arsenic trioxide induced cardiac fibrosis by inhibiting endothelial mesenchymal transition.

BACKGROUD: Arsenic trioxide (ATO), the first-line drug in treating acute premyelogenous leukemia, has the profound side effect of inducing endothelial mesenchymal transition (EndMT) and causing cardiac fibrosis. Diosgenin (DIO), a pharmaceutical compound found in Paris polyphylla, exhibits promising potential in safeguarding cardiovascular health by mitigating EndMT. PURPOSE: This study aims to explore the role and mechanism of DIO in ATO-induced myocardial fibrosis to provide a novel therapeutic agent for ATO-induced cardiac fibrosis. METHODS: Wistar rats were given DIO by gavage and ATO by tail vein. Cardiac function and fibrosis were evaluated by echocardiography and Masson's trichrome staining in rats. Human aortic endothelial cells (HAECs) were utilized to analyze ATO-induced EndMT in vitro. The cytoskeleton of HAECs was visualized using F-actin staining to observe cell morphology, while Dil-Ac-LDL staining was employed to assess cell functionality. EndMT-related factors (CD31 and α-SMA), glucocorticoid receptor (GR) and interleukin-6 (IL-6) were detected by immunofluorescence and Western blot in vivo and in vitro. Furthermore, GR was knocked down by si-GR, and IL-6 was blocked by IL-6 neutralizing antibody to verify their role in the effect of DIO on ATO-induced EndMT in HAECs. RESULTS: DIO exhibited significant efficacy in ATO-induced damage to both cardiac diastolic and systolic function, along with mitigating cardiac fibrosis. Additionally, DIO alleviated the loss of cytoskeletal anisotropy and enhanced the uptake of Dil-Ac-LDL in HAECs. Furthermore, it reversed the ATO-induced downregulation of endothelial-specific markers CD31 and GR, while suppressing the upregulation of mesenchymal markers α-SMA and IL-6, both in vivo and in vitro. Notably, the protective effect of DIO was compromised upon knockdown of GR, which also led to a reversal of DIO-induced IL-6 downregulation. Furthermore, the neutralization of IL-6 with specific antibodies abolished the ATO-induced changes related to EndMT. CONCLUSION: In this study, we clarified the protective effect of DIO on ATO-induced myocardial fibrosis against EndMT via the GR/IL-6 axis for the first time and provided a potential therapeutic agent for preventing heart damage caused by ATO.

Effects of Nox4 upregulation on PECAM-1 expression in a mouse model of diabetic retinopathy.

Diabetic Retinopathy (DR) is the leading cause of vision loss in working-age adults. The hallmark features of DR include vascular leakage, capillary loss, retinal ischemia, and aberrant neovascularization. Although the pathophysiology is not fully understood, accumulating evidence supports elevated reactive oxygen species associated with increased activity of NADPH oxidase 4 (Nox4) as major drivers of disease progression. Previously, we have shown that Nox4 upregulation in retinal endothelial cells by diabetes leads to increased vascular leakage by an unknown mechanism. Platelet endothelial cell adhesion molecule 1 (PECAM-1) is a cell surface molecule that is highly expressed in endothelial cells and regulates endothelial barrier function. In the present study, using endothelial cell-specific human Nox4 transgenic (TG) mice and endothelial cell-specific Nox4 conditional knockout (cKO) mice, we investigated the impact of Nox4 upregulation on PECAM-1 expression in mouse retinas and brain microvascular endothelial cells (BMECs). Additionally, cultured human retinal endothelial cells (HRECs) transduced with adenovirus overexpressing human Nox4 were used in the study. We found that overexpression of Nox4 increases PECAM-1 mRNA but has no effect on its protein expression in the mouse retina, BMECs, or HRECs. Furthermore, PECAM-1 mRNA and protein expression was unchanged in BMECs isolated from cKO mice compared to wild type (WT) mice with or without 2 months of diabetes. Together, these findings do not support a significant role of Nox4 in the regulation of PECAM-1 expression in the diabetic retina and endothelial cells. Further studies are warranted to elucidate the mechanism of Nox4-induced vascular leakage by investigating other intercellular junctional proteins in endothelial cells and their implications in the pathophysiology of diabetic retinopathy.