[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.

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.

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.

[α2-macroglobulin alleviates glucocorticoid-induced avascular necrosis of the femoral head in mice by promoting proliferation, migration and angiogenesis of vascular endothelial cells].

OBJECTIVE: To explore the mechanism underlying the protective effect of α2-macroglobulin (A2M) against glucocorticoid-induced femoral head necrosis. METHODS: In a human umbilical vein endothelial cell (HUVEC) model with injuries induced by gradient concentrations of dexamethasone (DEX; 10-8-10-5 mol/L), the protective effects of A2M at 0.05 and 0.1 mg/mL were assessed by examining the changes in cell viability, migration, and capacity of angiogenesis using CCK-8 assay, Transwell and scratch healing assays and angiogenesis assay. The expressions of CD31 and VEGF-A proteins in the treated cells were detected using Western blotting. In BALB/c mouse models of avascular necrosis of the femoral head induced by intramuscular injections of methylprednisolone, the effects of intervention with A2M on femoral trabecular structure, histopathological characteristics, and CD31 expression were examined with Micro-CT, HE staining and immunohistochemical staining. RESULTS: In cultured HUVECs, DEX treatment significantly reduced cell viability, migration and angiogenic ability in a concentration- and time-dependent manner (P<0.05), and these changes were obviously reversed by treatment with A2M in positive correlation with A2M concentration (P<0.05). DEX significantly reduced the expression of CD31 and VEGF-A proteins in HUVECs, while treatment with A2M restored CD31 and VEGF-A expressions in the cells (P<0.05). The mouse models of femoral head necrosis showed obvious trabecular damages in the femoral head, where a large number of empty lacunae and hypertrophic fat cells could be seen and CD31 expression was significantly decreased (P<0.05). A2M treatment of the mouse models significantly improved trabecular damages, maintained normal bone tissue structures, and increased CD31 expression in the femoral head (P<0.05). CONCLUSION: A2M promotes proliferation, migration, and angiogenesis of DEX-treated HUVECs and alleviates methylprednisolone-induced femoral head necrosis by improving microcirculation damages and maintaining microcirculation stability in the femoral head.

[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.

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.

Gintonin Alleviates HCl/Ethanol- and Indomethacin-Induced Gastric Ulcers in Mice.

Gintonin, newly extracted from ginseng, is a glycoprotein that acts as an exogenous lysophosphatidic acid (LPA) receptor ligand. This study aimed to demonstrate the in vivo preventive effects of gintonin on gastric damage. ICR mice were randomly assigned to five groups: a normal group (received saline, 0.1 mL/10 g, p.o.); a control group (administered 0.3 M HCl/ethanol, 0.1 mL/10 g, p.o.) or indomethacin (30 mg/kg, p.o.); gintonin at two different doses (50 mg/kg or 100 mg/kg, p.o.) with either 0.3 M HCl/ethanol or indomethacin; and a positive control (Ranitidine, 40 mg/kg, p.o.). After gastric ulcer induction, the gastric tissue was examined to calculate the ulcer index. The expression of gastric damage markers, such as tumor necrosis factor (TNF)-α, cyclooxygenase 2 (COX-2), and LPA2 and LPA5 receptors, were measured by Western blotting. Interleukin-6 (IL-6) and prostaglandin E2 (PGE2) levels were measured by enzyme-linked immunosorbent assay. The platelet endothelial cell adhesion molecule (PECAM-1), Evans blue, and occludin levels in gastric tissues were measured using immunofluorescence analysis. Both HCl/ethanol- and indomethacin-induced gastric ulcers showed increased TNF-α, IL-6, Evans blue permeation, and PECAM-1, and decreased COX-2, PGE2, occludin, and LPA5 receptor expression levels. However, oral administration of gintonin alleviated the gastric ulcer index induced by HCl/ethanol and indomethacin in a dose-dependent manner. Gintonin suppressed TNF-α and IL-6 expression, but increased COX-2 expression and PGE2 levels in mouse gastric tissues. Gintonin intake also increased LPA5 receptor expression in mouse gastric tissues. These results indicate that gintonin can play a role in gastric protection against gastric damage induced by HCl/ethanol or indomethacin.

PECAM-1 mediates temsirolimus-induced increase in neutrophil transendothelial migration that leads to lung injury.

Temsirolimus is a first-generation mTOR inhibitor commonly used in the clinical treatment of cancers that is associated with lung injury. However, the mechanism underlying this adverse effect remains elusive. Endothelial barrier dysfunction plays a pivotal role in the infiltration of neutrophils into the pulmonary alveoli, which eventually induces lung injury. The present study demonstrates that temsirolimus induces the aberrant expression of adhesion molecules in endothelial cells, leading to enhanced neutrophil infiltration and subsequent lung injury. Results of a mouse model revealed that temsirolimus disrupted capillary-alveolar barrier function and facilitated neutrophil transmigration across the endothelium within the alveolar space. Consistent with our in vivo observations, temsirolimus impaired intercellular barrier function within monolayers of human lung endothelial cells, resulting in increased neutrophil infiltration. Furthermore, we demonstrated that temsirolimus-induced neutrophil transendothelial migration was mediated by platelet endothelial cell adhesion molecule-1 (PECAM-1) in both in vitro and in vivo experiments. Collectively, these findings highlight that temsirolimus induces endothelial barrier dysfunction via PECAM-1-dependent pathway both in vitro and in vivo, ultimately leading to neutrophil infiltration and subsequent pulmonary injury.

Mechanotransduction via endothelial adhesion molecule CD31 initiates transmigration and reveals a role for VEGFR2 in diapedesis.

Engagement of platelet endothelial cell adhesion molecule 1 (PECAM, PECAM-1, CD31) on the leukocyte pseudopod with PECAM at the endothelial cell border initiates transendothelial migration (TEM, diapedesis). We show, using fluorescence lifetime imaging microscopy (FLIM), that physical traction on endothelial PECAM during TEM initiated the endothelial signaling pathway. In this role, endothelial PECAM acted as part of a mechanotransduction complex with VE-cadherin and vascular endothelial growth factor receptor 2 (VEGFR2), and this predicted that VEGFR2 was required for efficient TEM. We show that TEM required both VEGFR2 and the ability of its Y1175 to be phosphorylated, but not VEGF or VEGFR2 endogenous kinase activity. Using inducible endothelial-specific VEGFR2-deficient mice, we show in three mouse models of inflammation that the absence of endothelial VEGFR2 significantly (by ≥75%) reduced neutrophil extravasation by selectively blocking diapedesis. These findings provide a more complete understanding of the process of transmigration and identify several potential anti-inflammatory targets.

PECAM-1 drives ß-catenin-mediated EndMT via internalization in colon cancer with diabetes mellitus.

BACKGROUND: Diabetes mellitus (DM) is considered to be a risk factor in carcinogenesis and progression, although the biological mechanisms are not well understood. Here we demonstrate that platelet-endothelial cell adhesion molecule 1 (PECAM-1) internalization drives ß-catenin-mediated endothelial-mesenchymal transition (EndMT) to link DM to cancer. METHODS: The tumor microenvironment (TME) was investigated for differences between colon cancer with and without DM by mRNA-microarray analysis. The effect of DM on colon cancer was determined in clinical patients and animal models. Furthermore, EndMT, PECAM-1 and Akt/GSK-3ß/ß-catenin signaling were analyzed under high glucose (HG) and human colon cancer cell (HCCC) supernatant (SN) or coculture conditions by western and immunofluorescence tests. RESULTS: DM promoted the progression and EndMT occurrence of colon cancer (CC). Regarding the mechanism, DM induced PECAM-1 defection from the cytomembrane, internalization and subsequent accumulation around the cell nucleus in endothelial cells, which promoted ß-catenin entry into the nucleus, leading to EndMT occurrence in CC with DM. Additionally, Akt/GSK-3ß signaling was enhanced to inhibit the degradation of ß-catenin, which regulates the process of EndMT. CONCLUSIONS: PECAM-1 defects and/or internalization are key events for ß-catenin-mediated EndMT, which is significantly boosted by enhanced Akt/GSK-3ß signaling in the DM-associated TME. This contributes to the mechanism by which DM promotes the carcinogenesis and progression of CC. Video Abstract.

E2F2 modulates cell adhesion through the transcriptional regulation of PECAM1 in multiple myeloma.

Multiple myeloma (MM) is the second most common haematological malignancy. Despite the development of new drugs and treatments in recent years, the therapeutic outcomes of patients are not satisfactory. It is necessary to further investigate the molecular mechanism underlying MM progression. Herein, we found that high E2F2 expression was correlated with poor overall survival and advanced clinical stages in MM patients. Gain- and loss-of-function studies showed that E2F2 inhibited cell adhesion and consequently activated cell epithelial-to-mesenchymal transition (EMT) and migration. Further experiments revealed that E2F2 interacted with the PECAM1 promoter to suppress its transcriptional activity. The E2F2-knockdown-mediated promotion of cell adhesion was significantly reversed by the repression of PECAM1 expression. Finally, we observed that silencing E2F2 significantly inhibited viability and tumour progression in MM cell models and xenograft mouse models respectively. This study demonstrates that E2F2 plays a vital role as a tumour accelerator by inhibiting PECAM1-dependent cell adhesion and accelerating MM cell proliferation. Therefore, E2F2 may serve as a potential independent prognostic marker and therapeutic target for MM.

Porphyromonas gingivalis Gingipains Destroy the Vascular Barrier and Reduce CD99 and CD99L2 Expression To Regulate Transendothelial Migration.

Porphyromonas gingivalis is an important periodontal pathogen that can cause vascular injury and invade local tissues through the blood circulation, and its ability to evade leukocyte killing is critical to its distal colonization and survival. Transendothelial migration (TEM) is a series of that enable leukocytes to squeeze through endothelial barriers and migrate into local tissues to perform immune functions. Several studies have shown that P. gingivalis-mediated endothelial damage initiates a series of proinflammatory signals that promote leukocyte adhesion. However, whether P. gingivalis is involved in TEM and thus influences immune cell recruitment remains unknown. In our study, we found that P. gingivalis gingipains could increase vascular permeability and promote Escherichia coli penetration by downregulating platelet/endothelial cell adhesion molecule 1 (PECAM-1) expression in vitro. Furthermore, we demonstrated that although P. gingivalis infection promoted monocyte adhesion, the TEM capacity of monocytes was substantially impaired, which might be due to the reduced CD99 and CD99L2 expression on gingipain-stimulated endothelial cells and leukocytes. Mechanistically, gingipains mediate CD99 and CD99L2 downregulation, possibly through the inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway. In addition, our in vivo model confirmed the role of P. gingivalis in promoting vascular permeability and bacterial colonization in the liver, kidney, spleen, and lung and in downregulating PECAM-1, CD99, and CD99L2 expression in endothelial cells and leukocytes. IMPORTANCE P. gingivalis is associated with a variety of systemic diseases and colonizes in distal locations in the body. Here, we found that P. gingivalis gingipains degrade PECAM-1 to promote bacterial penetration while simultaneously reducing leukocyte TEM capacity. A similar phenomenon was also observed in a mouse model. These findings established P. gingivalis gingipains as the key virulence factor in modulating the permeability of the vascular barrier and TEM processes, which may provide a new rationale for the distal colonization of P. gingivalis and its associated systemic diseases.

Gemcitabine alters sialic acid binding of the glycocalyx and induces inflammatory cytokine production in cultured endothelial cells.

Gemcitabine (GEM) is an anticancer drug inhibiting DNA synthesis. Glomerular thrombotic microangiopathy (TMA) has been reported as an adverse effect. However, the precise mechanism of GEM-induced endothelial injury remains unknown. Cultured human umbilical vein endothelial cells (HUVECs) in the confluent phase were exposed to GEM (5-100 µM) for 48 h and evaluated cell viability and morphology, lectin binding concerning sialic acid of endothelial glycocalyx (GCX), and immunofluorescent staining of platelet-endothelial cell adhesion molecule (PECAM) and vascular endothelial growth factor receptor 2 (VEGFR2). The mRNA expression of α2,6-sialyltransferase (ST6Gal1), sialidase (neuraminidase-1: NEU-1), and interleukin (IL)-1ß and IL-6 was also evaluated. GEM exposure at 5 µM induced cellular shrinkage and intercellular dissociation, accompanied by slight attenuation of PECAM and VEGFR2 immunostaining, although cell viability was still preserved. At this concentration, lectin binding showed a reduction of terminal sialic acids in endothelial GCX, probably associated with reduced ST6Gal1 mRNA expression. IL-1ß and IL-6 mRNA expression was significantly increased after GEM exposure. GEM reduced terminal sialic acids in endothelial GCX through mRNA suppression of ST6Gal1 and induced inflammatory cytokine production in HUVECs. This phenomenon could be associated with the mechanism of GEM-induced TMA.

Angiogenetic transcriptional profiling reveals potential targets modulated in blood of patients with cardiovascular disorders.

OBJECTIVES: Based on the angiogenetic, transcriptional profile of non-diseased and arteriosclerotic vessels, we aim to identify the leucocytic markers as a potential, minimal invasive tool supporting diagnosis of vascular pathology. METHODS: Transcriptional profiling was performed with Angiogenesis RT2 Profiler PCR (Polymerase Chain Reaction) array on three non-pathological and three arteriosclerotic vessels, followed by immunohistochemical staining. Based on these screening results, selected transcripts were employed for qPCR with specific primers and investigated on the blood RNA (RiboNucleic Acid) obtained from nine healthy controls and 29 patients with cardiovascular disorders. Thereafter, expression of these transcripts was investigated in vitro in human monocytes under calcification-mimicking conditions. RESULTS AND CONCLUSIONS: Transcriptional profiling on the vessels revealed that out of 84 targets investigated two were up-regulated more than 100-fold, 18 more than 30 and 15 more than 10, while the most noticeable down-regulation was observed by ephrin-A3 and platelet-derived growth factor alpha (PDGFA) genes. Based on the vessel results, investigations of the selected blood transcripts revealed that thrombospondin 1 (THBS1), thrombospondin 3 (THBS3), transforming growth factor, beta receptor 1 (TGFBR1), platelet-derived growth factor alpha, plasminogen activator, urokinase (PLAU) and platelet/endothelial cell adhesion molecule 1 (PECAM-1) were significantly elevated in cardiovascular blood as compared to corresponding controls. Induction of calcification-related conditions in vitro to human THP-1 monocytes led to noticeable modulation of these transcripts. Taken together, these data demonstrate that leucocytic THBS1, THBS3, TGFBR1, platelet-derived growth factor alpha, PLAU and PECAM-1 have a correlation with cardiovascular disorders and could be used as a supportive tool predicting development of this pathological condition.

The Beneficial Impact of Zinc Supplementation on the Vascular Tissue of the Abdominal Aorta under Repeated Intoxication with Cadmium: A Study in an In Vivo Experimental Model.

In an in vivo rat model of human exposure to cadmium (Cd; 5 and 50 mg/L, 6 months), whether the supplementation with zinc (Zn; 30 and 60 mg/L, increasing its daily intake by 79% and 151%, respectively) protects against the unfavourable impact of this xenobiotic on the vascular tissue of the abdominal aorta was investigated. The treatment with Cd led to oxidative stress and increased the concentrations of pro-inflammatory interleukin 1ß (IL-1ß), total cholesterol (TC), triglycerides (TG), and endothelial nitric oxide synthase (eNOS) and decreased the concentration of anti-inflammatory interleukin 10 (IL-10) in the vascular tissue. Cd decreased the expression of intercellular adhesion molecule-1 (ICAM-1), platelet endothelial cell adhesion molecule-1 (PECAM-1), and L-selectin on the endothelial cells. The administration of Zn prevented most of the Cd-induced alterations or at least weakened them (except for the expression of adhesive molecules). In conclusion, Zn supplementation may protect from the toxic impact of Cd on the blood vessels and thus exert a beneficial influence on the cardiovascular system. The increase in the intake of Zn by 79% may be sufficient to provide this protection and the effect is related to the antioxidative, anti-inflammatory, and antiatherogenic properties of this essential element.

Neutrophil depletion in the pre-implantation phase impairs pregnancy index, placenta and fetus development.

Maternal neutrophils cells are players in gestational tolerance and fetus delivery. Nonetheless, their actions in each phase of the pregnancy are unknown. We here investigated the role of maternal neutrophil depletion before the blastocyst implantation phase and outcomes in the pregnancy index, placenta, and fetus development. Neutrophils were pharmacologically depleted by i.p. injection of anti-Gr1 (anti-neutrophils; 200 µg) 24 hours after plug visualization in allogeneic-mated C57BL/6/BALB/c mice. Depletion of peripheral neutrophils lasted until 48 hours after anti-Gr1 injection (gestational day 1.5-3.5). On gestational day 5.5, neutrophil depletion impaired the blastocyst implantation, as 50% of pregnant mice presented reduced implantation sites. On gestational day 18.5, neutrophil depletion reduced the pregnancy rate and index, altered the placenta disposition in the uterine horns, and modified the structure of the placenta, detected by reduced junctional zone, associated with decreased numbers of giant trophoblast cells, spongiotrophoblast. Reduced number of placenta cells labeled for vascular endothelial growth factor (VEGF), platelet-endothelial cell adhesion molecule (PECAM-1), and intercellular cell adhesion molecule (ICAM-1), important markers of angiogenesis and adhesiveness, were detected in neutrophil depleted mice. Furthermore, neutrophil depletion promoted a higher frequency of monocytes, natural killers, and T regulatory cells, and lower frequency of cytotoxic T cells in the blood, and abnormal development of offspring. Associated data obtained herein highlight the pivotal role of neutrophils actions in the early stages of pregnancy, and address further investigations on the imbricating signaling evoked by neutrophils in the trophoblastic interaction with uterine epithelium.

Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells Accelerate Diabetic Wound Healing via Promoting M2 Macrophage Polarization, Angiogenesis, and Collagen Deposition.

Some scholars have suggested that the clinical application of exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs-exo) might represent a novel strategy to improve diabetic wound healing. However, the mechanisms underlying the effects of hucMSCs-exo on wound healing remain poorly understood. This study aimed to identify the mechanism of hucMSCs-exo in treating diabetic wounds. HucMSCs-exo were isolated from human umbilical cord mesenchymal stem cells (hucMSCs) and subcutaneously injected into full-thickness wounds in diabetic rats. Wound healing closure rates and histological analysis were performed. The levels of tumor necrosis factor-α (TNF-α), macrophage mannose receptor (MMR/CD206), platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), and vascular endothelial growth factor (VEGF) were detected by immunohistochemistry. The degree of collagen deposition was examined using Masson's trichrome staining. Gross evaluation of wound healing was carried out from day 0 to 14 post-surgery, and the wound site was harvested for histology on days 3, 7, and 14 post-wounding. HucMSCs-exo transplantation increased diabetic wound healing. In vitro, hucMSCs-exo promoted the proliferation of human umbilical vein endothelial cells (HUVECs) and NIH-3T3 cells. In vivo, hucMSCs-exo reduced wound area and inflammatory infiltration and increased collagen fibers. In addition, wound tissues in the hucMSCs-exo group had higher CD206, CD31, and VEGF expressions and lower TNF-α levels than those in the control group on day 14. Our results demonstrated that hucMSCs-exo facilitated diabetic wound repair by inducing anti-inflammatory macrophages and promoting angiogenesis and collagen deposition.

[Impaired production of platelet-endothelial cell adhesion molecules as an early diagnostic marker of heart transplant rejection]. / Narushenie produktsii molekul trombotsitarno-endotelial'noi kletochnoi adgezii kak rannii diagnosticheskii marker ottorzheniya serdechnogo transplantata.

OBJECTIVE: Reveal the involvement of platelet-endothelial cell adhesion molecules PECAM-1 in the transplanted heart rejection pathogenesis and determine the CD31 marker significance for biopsy diagnostics of the process form and degree. MATERIAL AND METHODS: Sections from endomyocardial biopsies of 56 heart transplant recipients were stained with hematoxylin and eosin. The streptavidin-biotin method was used to determine the expression of T-lymphocytes (CD3), B-lymphocytes (CD20), macrophages (CD68) and the C4d component of complement to determine the form and degree of graft rejection. Additionally, the expression of platelet-endothelial cell adhesion molecules PECAM-1 (CD31) was detected. Using computer morphometry in digital images, the area of pathological changes and the area of CD31 expression was measured with the calculation of the staining area coefficient. RESULTS: The highest levels of PECAM-1 expression were found in the absence of a heart transplant rejection. The degree of rejection of 1R is characterized by a decrease in expression by 1.3 times, when there are no significant signs of necrosis in the myocardium, the area of which increases sharply at degree 2R by 163.7 times, and at 3R by 570.7 times compared with 1R. The process proceeds in parallel with a further decrease in the level of CD31 expression and is accompanied by the development of hemorrhagic manifestations. The intensity of hemorrhages in the myocardium increases by 7.3 times with grade 3R compared with 1R. CONCLUSION: Expression of PECAM-1 reflects the state of the vascular bed of the heart transplant. Its decrease can be considered as an early pathomorphological marker of transplanted heart rejection. The expression of CD31 continues to decrease with increasing severity of rejection and is accompanied by the progressive development of necrosis and hemorrhages in the graft heart muscle.

Immunohistochemical analysis of CD31 expression in myocardial tissues from autopsies of patients with ischemic heart disease.

CD31, a transmembrane protein expressed on endothelial and hematopoietic cells, plays important roles in leukocyte trafficking, mechanotransduction, angiogenesis, vascular permeability, and regulation of cellular responsiveness. CD31 immunoreactivity is employed as a sensitive and specific endothelial marker in diagnostic pathology. In this study, CD31 expression in myocardial tissues from deceased patients with ischemic heart disease and a mouse model of acute myocardial infarction were examined by immunohistochemical staining. We examined 24 neutral formalin-fixed, paraffin-embedded myocardial tissue samples obtained within 48 h postmortem from the autopsies of patients who were diagnosed with ischemic heart disease. CD31 expression was observed in vascular endothelial and endocardial cells. In necrotic myocardium, diffusion of CD31 antigen was observed. Elevated CD31 expression was observed around myocardial cells undergoing remodeling, suggesting that endothelial proliferation occurred at these sites. In contrast, fibrotic myocardial foci did not show upregulated CD31 expression. The same CD31 expression characteristics as those observed in the human samples were observed in the mouse model. CD31 immunostaining as an endothelial and microvasculature marker may be a useful complement to conventional staining techniques currently used in the diagnosis of ischemic heart disease, and may allow the timing and process of myocardial remodeling to be analyzed in detail.