Normal Vascular Tissue Research Articles

Simultaneously blocking ANGPTL3 and CD47 prevents the progression of atherosclerosis through regulating lipid metabolism, macrophagic efferocytosis and lipid peroxidation

Atherosclerosis (AS) ultimately cause major adverse cardiovascular events (MACEs). While traditional strategies by lipid-reducing have reduced MACEs, many patients continue to face significant risks. It might attribute to the upregulation of CD47 expression in AS lesions, that mediated anti-efferocytosis of macrophages. Therefore, we propose simultaneously blocking ANGPTL3, a vital regulator of lipid metabolism, and CD47 might be a potential approach for AS therapy. Firstly, we investigate the role of a novel anti-ANGPTL3 nanobody-Fc (FD03) in AS. We found that FD03 treatment significantly decreased circulating lipids, plaque size, and lipid deposition in apoE-/- mice compared to control Ab, but there was a twofold increase in plaque formation in comparison to baseline. However, immunofluorescence indicated the upregulation of CD47 expression in the plaques even after FD03 treatment compared to normal vascular tissue. Next, a bifunctional protein containing signal regulatory protein alpha (SIRPα) and FD03 (SIRPαD1-FD03) was constructed to block CD47 and ANGPTL3 concurrently, which had high purity, robust stability, and high affinity to CD47 and ANGPTL3 with biological activity in vitro. Furthermore, SIRPαD1-FD03 fusion protein exhibited the enhanced therapeutic effect on AS compared with SIRPαD1-Fc or FD03, regressing plaque contents and the necrotic core equal to baseline. Mechanistically, SIRPαD1-FD03 reduced serum lipids, augmented the efferocytosis rate and macrophage M2 polarization, and decreased the reactive oxygen species (ROS) and lipid peroxidation level in atherosclerotic plaques. Collectively, our project suggests an effective approach for AS by simultaneously blocking ANGPTL3 and CD47 to regulate lipid metabolism, macrophage activity and lipid peroxidation.

Inhibiting YAP1 reduced abdominal aortic aneurysm formation by suppressing adventitial fibroblast phenotype transformation and migration.

The adventitial fibroblast (AF) is the most abundant cell in the vascular adventitia, a few studies had confirmed that AF contributed to abdominal aortic aneurysm (AAA) development; YAP1 involved in several vascular diseases by promoting AF transformed to myofibroblast, the role of YAP1 in AAA is not clear yet. This study aims to determine whether YAP1 play a role in AAA process by regulating AF function. We found the expression of YAP1was significantly increased in aneurysm tissues of AAA patients compared to normal adjacent vascular tissues and mainly in adventitia. YAP1 also upregulated in elastase-induced and CaCl2-induced mice AAA model. Suppressed YAP1 function with YAP1 inhibitor-Verteporfin declined AAA incident rate remarkably in mice, and the collagen deposition in the adventitia was alleviated obviously. Afterwards, we studied the effect of YAP1 on the function of AF, Verteporfin was used to block YAP1 invitro, the process of AF transforming to myofibroblast and migration were almost completely eliminated after inhibiting YAP1 expression. This study demonstrated that YAP1 may play a key role in AAA development, inhibiting YAP1 significantly reduced AAA formation through suppressed the process of AF transformed to myofibroblast and migration.

CD6 and CCR7 as Genetic Biomarkers in Evaluating Intracranial Aneurysm Rupture Risk.

This study used bioinformatics combined with statistical methods to identify plasma biomarkers that can predict intracranial aneurysm (IA) rupture and provide a strong theoretical basis for the search for new IA rupture prevention methods. We downloaded gene expression profiles in the GSE36791 and GSE122897 datasets from the Gene Expression Omnibus (GEO) database. Data were normalized using the "sva" R package and differentially expressed genes (DEGs) were identified using the "limma" R package. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were used for DEG function analysis. Univariate logistic regression analysis, least absolute shrinkage and selection operator (LASSO) regression modeling, and the support vector machine recursive feature elimination (SVM-RFE) algorithm were used to identify key biomarker genes. Data from GSE122897 and GSE13353 were extracted to verify our findings. Eight co-DEG mRNAs were identified in the GSE36791 and GSE122897 datasets. Genes associated with inflammatory responses were clustered in the co-DEG mRNAs in IAs. CD6 and C-C chemokine receptor 7 (CCR7) were identified as key genes associated with IA. CD6 and CCR7 were upregulated in patients with IA and their expression levels were positively correlated. There were significant differences in the infiltration of immune cells between IAs and normal vascular wall tissues (p < 0.05). A predictive nomogram was designed using this two-gene signature. Binary transformation of CD6 and CCR7 was performed according to the cut-off value to construct the receiver-operating characteristic (ROC) curve and showed a strong predictive ability of the CD6-CCR7 gene signature (p < 0.01; area under the curve (AUC): 0.90; 95% confidence interval (CI): 0.88-0.92). Furthermore, validation of this two-gene signature using the GSE122897 and GSE13353 datasets proved it to be valuable for clinical application. The identified two-gene signature (CD6-CCR7) for evaluating the risk of IA rupture demonstrated good clinical application value.

Differential roles of normal and lung cancer-associated fibroblasts in microvascular network formation

Perfusable microvascular networks offer promising three-dimensional in vitro models to study normal and compromised vascular tissues as well as phenomena such as cancer cell metastasis. Engineering of these microvascular networks generally involves the use of endothelial cells stabilized by fibroblasts to generate robust and stable vasculature. However, fibroblasts are highly heterogenous and may contribute variably to the microvascular structure. Here, we study the effect of normal and cancer-associated lung fibroblasts on the formation and function of perfusable microvascular networks. We examine the influence of cancer-associated fibroblasts on microvascular networks when cultured in direct (juxtacrine) and indirect (paracrine) contacts with endothelial cells, discovering a generative inhibition of microvasculature in juxtacrine co-cultures and a functional inhibition in paracrine co-cultures. Furthermore, we probed the secreted factors differential between cancer-associated fibroblasts and normal human lung fibroblasts, identifying several cytokines putatively influencing the resulting microvasculature morphology and functionality. These findings suggest the potential contribution of cancer-associated fibroblasts in aberrant microvasculature associated with tumors and the plausible application of such in vitro platforms in identifying new therapeutic targets and/or agents that can prevent formation of aberrant vascular structures.

Revealing PPP1R12B and COL1A1 as piRNA pathway genes contributing to abdominal aortic aneurysm through integrated analysis and experimental validation

BackgroundAbdominal aortic aneurysm (AAA) is a permanent dilation of the abdominal aorta, with a high mortality rate when rupturing. Although lots of piRNA pathway genes (piRPGs) have recently been linked to both neoplastic and non-neoplastic illnesses, their role in AAA is still unknown. Utilizing integrative bioinformatics methods, this research discovered piRPGs as biomarkers for AAA and explore possible molecular mechanisms. MethodsThe datasets were obtained from the Gene Expression Omnibus and piRPGs were identified from the Genecards database. The “limma” and “clusterProfiler” R-packages were used to discover differentially expressed genes and perform enrichment analysis, respectively. Hub piRPGs were further filtered using least absolute shrinkage and selection operator regression, random forests, as well as receiver operating characteristic curve. Additionally, multi-factor logistic regression (MLR), extreme gradient boosting (XGboost), and artificial neural network (ANN) were employed to construct prediction models. The relationship between hub piRPGs and immune infiltrating cells and sgGSEA were further studied. The expression of hub piRPGs was verified by qRT-PCR, immunohistochemistry, and western blotting in AAA and normal vascular tissues and analyzed by scRNA-seq in mouse AAA model. SRAMP and cMAP database were utilized for the prediction of N6-methyladenosine (m6A) targets therapeutic drug. Results34 differentially expressed piRPGs were identified in AAA and enriched in pathways of immune regulation and gene silence. Three piRPGs (PPP1R12B, LRP10, and COL1A1) were further screened as diagnostic genes and used to construct prediction model. Compared with MLR and ANN, Xgboost showed better predictive ability, and PPP1R12B might have the ability to distinguish small and large AAA. Furthermore, the expression levels of PPP1R12B and COL1A1 were consistent with the results of bioinformatics analysis, and PPP1R12B showed a downward trend that may be related to m6A. ConclusionThe results suggest that piRPGs might serve a significant role in AAA. PPP1R12B, COL1A1, and LRP10 had potential as diagnostic-specific biomarkers for AAA and performed better in XGboost model. The expression and localization of PPP1R12B and COL1A1 were experimentally verified. Besides, downregulation of PPP1R12B caused by m6A might contribute to the formation of AAA.

Effects of COL1A1 and SYTL2 on inflammatory cell infiltration and poor extracellular matrix remodeling of the vascular wall in thoracic aortic aneurysm.

Thoracic aortic aneurysm (TAA) is a fatal cardiovascular disease, the pathogenesis of which has not yet been clarified. This study aimed to identify and validate the diagnostic markers of TAA to provide a strong theoretical basis for developing new methods to prevent and treat this disease. Gene expression profiles of the GSE9106, GSE26155, and GSE155468 datasets were acquired from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified using the "limma" package in R. Least absolute shrinkage and selection operator (LASSO), support vector machine-recursive feature elimination (SVM-RFE), random forest, and binary logistic regression analyses were used to screen the diagnostic marker genes. Single-sample gene set enrichment analysis (ssGSEA) was used to estimate immune cell infiltration in TAA. A total of 16 DEGs were identified. The enrichment and functional correlation analyses showed that DEGs were mainly associated with inflammatory response pathways and collagen-related diseases. Collagen type I alpha 1 chain ( COL1A1 ) and synaptotagmin like 2 ( SYTL2 ) were identified as diagnostic marker genes with a high diagnostic value for TAA. The expression of COL1A1 and SYTL2 was considerably higher in TAA vascular wall tissues than in the corresponding normal tissues, and there were significant differences in the infiltration of immune cells between TAA and normal vascular wall tissues. Additionally, COL1A1 and SYTL2 expression were associated with the infiltration of immune cells in the vascular wall tissue. Single-cell analysis showed that COL1A1 in TAA was mainly derived from fibroblasts and SYTL2 mainly from cluster of differentiation (CD)8 + T cells. In addition, single-cell analysis indicated that fibroblasts and CD8 + T cells in TAA were significantly higher than those in normal arterial wall tissue. COL1A1 and SYTL2 may serve as diagnostic marker genes for TAA. The upregulation of SYTL2 and COL1A1 may be involved in the inflammatory infiltration of the vessel wall and poor extracellular matrix remodeling, promoting the progression of TAA.

Identification of necroptosis-related gene TRAF5 as potential target of diagnosing atherosclerosis and assessing its stability

BackgroundAtherosclerosis (AS) is a leading cause of morbidity and mortality in older patients and features progressive formation of plaques in vascular tissues. With the progression of atherosclerosis, plaque rupture may occur and cause stroke, myocardial infarction, etc. Different forms of cell death promote the formation of a necrotic core of the plaque, leading to rupture. Necroptosis is a type of programmed cell death that contributes to the development of cardiovascular disease. However, the role of necroptosis in AS has not yet been investigated.MethodsThe Gene Expression Omnibus (GEO) database was used to obtain gene expression profiles. Differentially expressed genes (DEGs) and necroptosis gene sets were used to identify necroptosis-related differentially expressed genes (NRDEGs). The NRDEGs were used to construct a diagnostic model and were further screened using least absolute shrinkage selection operator (LASSO) regression and random forest (RF) analysis. The discriminatory capacity of the NRDEGs was evaluated using receiver operating characteristic (ROC) curves. Immune infiltration levels were estimated based on CIBERSORTx analysis. The GSE21545 dataset, containing survival information, was used to determine prognosis-associated genes. Univariate and multivariate Cox regression analyses combined with survival analysis determined gene prognostic values. RNA and protein levels were detected by RT-qPCR and western blotting in arteriosclerosis obliterans(ASO) and normal vascular tissues. Vascular smooth muscle cells (VSMCs) were treated with oxidized low-density lipoprotein (ox-LDL) to develop cell models of advanced AS. The effects of protein knockdown on necroptosis were assessed by western blotting and flow cytometry. EdU and Cell Counting Kit-8 assays were used to examine cell proliferation.ResultsTNF Receptor Associated Factor 5 (TRAF5) was identified as a diagnostic marker for AS based on the AUC value in both the GSE20129 and GSE43292 datasets. According to differential expression analysis, LASSO regression analysis, RF analysis, univariate analysis, multivariate analysis, and gene-level survival analysis, TRAF5 was markedly associated with necroptosis in AS. Silencing TRAF5 promotes necroptosis and attenuates the proliferation of ox-LDL-induced cell models of advanced AS.ConclusionsThis study identified a diagnostic marker of necroptosis-related atherosclerosis, TRAF5, which can also be used to diagnose and assess atherosclerotic plaque stability. This novel finding has important implications in the diagnosis and assessment of plaque stability in atherosclerosis.

Role of UDP-glucose ceramide glucosyltransferase in venous malformation.

Venous malformation (VM) results from the abnormal growth of the vasculature; however, the detailed molecular mechanism remains unclear. As a glycosyltransferase, UDP-glucose ceramide glucosyltransferase (UGCG) is localized to the Golgi body and is a key enzyme in the first step of glycosphingolipid synthesis. Here, we aimed to explore the relationship between UGCG and the development of VM. First, investigations using RT-qPCR and Western blotting on the diseased vasculature of VM patients and normal vascular tissues revealed that UGCG expression was markedly elevated in the diseased vessels. Subsequently, immunofluorescence assay showed that UGCG was co-localized with CD31, an endothelial cell marker, in tissues from patients with VM and healthy subjects. Then, we established TIE2-L914F-mutant human umbilical vein endothelial cells (HUVECs) by lentivirus transfection. Next, Western blotting revealed that UGCG expression was considerably higher in HUVECsTIE2-L914F. In addition, we established a UGCG-overexpressing HUVECs line by plasmid transfection. With the CCK8 cell proliferation experiment, wound healing assay, and tube formation assay, we found that UGCG could promote the proliferation, migration, and tube formation activity of HUVECs, whereas the inhibition of UGCG could inhibit the proliferation, migration, and tube formation activity of HUVECsTIE2-L914F. Finally, Western blotting revealed that UGCG regulates the AKT/mTOR pathway in HUVECs. These data demonstrated that UGCG can affect the activity of vascular endothelial cells and regulate the AKT/mTOR signaling pathway; this is a potential mechanism underlying VM pathogenesis.

Mechanism of Notch3 signaling pathway regulating the differentiation of aortic dissection vascular stem cells into smooth muscle cells

To explore whether the differentiation of vascular stem cells (VSC) into smooth muscle cells (SMC) in aortic dissection (AD) is dysregulated, and to verify the role of Notch3 pathway in this process. Aortic tissues were obtained from AD patients undergoing aortic vascular replacement and heart transplant donors at Department of Cardiovascular Surgery, Guangdong Provincial People's Hospital Affiliated to Southern Medical University. VSC were isolated by enzymatic digestion and c-kit immunomagnetic beads. The cells were divided into normal donor-derived VSC group (Ctrl-VSC group) and AD-derived VSC group (AD-VSC group). The presence of VSC in the aortic adventitia was detected by immunohistochemical staining, and VSC was identified by stem cell function identification kit. The differentiation model of VSC into SMC established in vitro was induced by transforming growth factor-β1 (10 μg/L) for 7 days. They were divided into normal donor VSC-SMC group (Ctrl-VSC-SMC group), AD VSC-SMC group (AD-VSC-SMC group) and AD VSC-SMC+Notch3 inhibitor DAPT group (AD-VSC-SMC+DAPT group,DAPT 20 μmol/L was added during differentiation induction). The expression of contractile marker Calponin 1 (CNN1) in SMC derived from aortic media and VSC were detected by immunofluorescence staining. The protein expressions of contractile markers α-smooth muscle actin (α-SMA), CNN1 as well as Notch3 intracellular domain (NICD3) in SMC derived from aortic media and VSC were detected by Western blotting. Immunohistochemical staining showed there was a population of c-kit-positive VSC in the adventitia of aortic vessels, and VSC from both normal donors and AD patients had the ability to differentiate into adipocytes and chondrocytes. Compared with normal donor vascular tissue, the expressions of SMC markers α-SMA and CNN1 of tunica media contraction in AD were down-regulated (α-SMA/β-actin: 0.40±0.12 vs. 1.00±0.11, CNN1/β-actin: 0.78±0.07 vs. 1.00±0.14, both P < 0.05), while the protein expression of NICD3 was up-regulated (NICD3/GAPDH: 2.22±0.57 vs. 1.00±0.15, P < 0.05). Compared with Ctrl-VSC-SMC group, the expressions of contractile SMC markers α-SMA and CNN1 were down-regulated in AD-VSC-SMC group (α-SMA/β-actin: 0.35±0.13 vs. 1.00±0.20, CNN1/β-actin: 0.78±0.06 vs. 1.00±0.07, both P < 0.05), the protein expression of NICD3 was up-regulated (NICD3/GAPDH: 22.32±1.22 vs. 1.00±0.06, P < 0.01). Compared with AD-VSC-SMC group, the expressions of contractile SMC markers α-SMA, CNN1 were up-regulated in AD-VSC-SMC+DAPT group (α-SMA/β-actin: 1.70±0.07 vs. 1.00±0.15, CNN1/β-actin: 1.62±0.03 vs. 1.00±0.02, both P < 0.05). Dysregulation of VSC differentiation into SMC occurs in AD, while inhibition of Notch3 pathway activation can restore the expression of contractile proteins in VSC-derived SMC in AD.

Identification of potential biomarkers of vascular calcification using bioinformatics analysis and validation in vivo.

BackgroundVascular calcification (VC) is the most widespread pathological change in diseases of the vascular system. However, we know poorly about the molecular mechanisms and effective therapeutic approaches of VC.MethodsThe VC dataset, GSE146638, was downloaded from the Gene Expression Omnibus (GEO) database. Using the edgeR package to screen Differentially expressed genes (DEGs). Gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) were used to find pathways affecting VC. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed on the DEGs. Meanwhile, using the String database and Cytoscape software to construct protein-protein interaction (PPI) networks and identify hub genes with the highest module scores. Correlation analysis was performed for hub genes. Receiver operating characteristic (ROC) curves, expression level analysis, GSEA, and subcellular localization were performed for each hub gene. Expression of hub genes in normal and calcified vascular tissues was verified by quantitative reverse transcription PCR (RT-qPCR) and immunohistochemistry (IHC) experiments. The hub gene-related miRNA-mRNA and TF-mRNA networks were constructed and functionally enriched for analysis. Finally, the DGIdb database was utilized to search for alternative drugs targeting VC hub genes.ResultsBy comparing the genes with normal vessels, there were 64 DEGs in mildly calcified vessels and 650 DEGs in severely calcified vessels. Spp1, Sost, Col1a1, Fn1, and Ibsp were central in the progression of the entire VC by the MCODE plug-in. These hub genes are primarily enriched in ossification, extracellular matrix, and ECM-receptor interactions. Expression level results showed that Spp1, Sost, Ibsp, and Fn1 were significantly highly expressed in VC, and Col1a1 was incredibly low. RT-qPCR and IHC validation results were consistent with bioinformatic analysis. We found multiple pathways of hub genes acting in VC and identified 16 targeting drugs.ConclusionsThis study perfected the molecular regulatory mechanism of VC. Our results indicated that Spp1, Sost, Col1a1, Fn1, and Ibsp could be potential novel biomarkers for VC and promising therapeutic targets.

Self-Tolerance of Vascular Tissues Is Broken Down by Vascular Dendritic Cells in Response to Systemic Inflammation to Initiate Regional Autoinflammation.

The correlation of infections with vascular autoinflammatory diseases such as vasculitis and atherosclerosis has been long recognized, and progressive inflammation with the formation of tertiary lymphoid organs in arterial adventitia intensively studied, the immunological basis of the nondiseased vasculatures that predispose to subsequent vascular autoimmunity during inflammation, however, is not well characterized. Here, we investigated the vascular immunity in situ of steady-state C57BL/6 mice and found that healthy vascular tissues contained a comprehensive set of immune cells with relatively higher proportion of innate components than lymphoid organs. Notably, a complete set of dendritic cell (DC) subsets was observed with monocyte-derived DCs (moDCs) constituting a major proportion; this is in contrast to moDCs being considered rare in the steady state. Interestingly, these vascular DCs constitutively expressed more suppressive factors with cDC1 for PD-L1 and moDCs for IL-10; this is concordant with the inhibitive phenotype of T cells in normal vascular tissues. The immunotolerant state of the vascular tissues, however, was readily eroded by systemic inflammation, demonstrated by the upregulation of proinflammatory cytokines and enhanced antigen presentation by vascular DCs to activate both cellular and humoral immunity in situ, which ultimately led to vascular destruction. Different vascular DC subsets elicited selective effects: moDCs were potent cytokine producers and B-cell activators, whereas cDCs, particularly, cDC1, were efficient at presenting antigens to stimulate T cells. Together, we unveil regional immunological features of vascular tissues to explain their dual facets under physiological versus pathological conditions for the better understanding and treatment of cardiovascular autoinflammation.

The role of miR-26a, miR-29a and miR-448-3p in the development of cerebral aneurysm.

Cerebral aneurysms cause the weakening and abnormal expansion of the arterial wall. MicroRNAs (miRNAs) are small noncoding RNAs of approximately 22 nucleotides in length that regulate gene expression. This study aimed to elucidate the role of miRNAs in the development of cerebral aneurysms. This study compared the expression levels of miR-26a, miR-29a and miR-448-3p in 50 samples each of cerebral aneurysm tissues and normal superficial temporal artery tissues. The miRNA expression levels were also compared in terms of aneurysm location and rupture status, i.e., presence or absence of rupture. Expression levels of miR-26a, miR-29a and miR-448-3p were increased in aneurysm tissues compared with normal vascular tissues. No significant difference was found in the miRNA expression levels with respect to aneurysm location or rupture status. This study showed that miR-26a, miR-29a and miR-448-3p overexpression could play an important role in intracranial aneurysm development independent of aneurysm location and rupture status. miR-26a, miR-29a and miR-448-3p could act as potential therapeutic targets in patients with intracranial aneurysms; however, further studies are needed on this issue.

Combining Bioinformatics Techniques to Study the Key Immune-Related Genes in Abdominal Aortic Aneurysm

Approximately 13,000 people die of an abdominal aortic aneurysm (AAA) every year. This study aimed to identify the immune response-related genes that play important roles in AAA using bioinformatics approaches. We downloaded the GSE57691 and GSE98278 datasets related to AAA from the Gene Expression Omnibus database, which included 80 AAA and 10 normal vascular samples. CIBERSORT was used to analyze the samples and detect the infiltration of 22 types of immune cells and their differences and correlations. The principal component analysis showed significant differences in the infiltration of immune cells between normal vascular and AAA samples. High proportions of CD4+ T cells, activated mast cells, resting natural killer cells, and 12 other types of immune cells were found in normal vascular tissues, whereas high proportions of macrophages, CD8+ T cells, resting mast cells, and six other types of immune cells were found in AAA tissues. In the selected samples, we identified 39 upregulated (involved in growth factor activity, hormone receptor binding, and cytokine receptor activity) and 133 downregulated genes (involved in T cell activation, cell chemotaxis, and regulation of immune response mediators). The key differentially expressed immune response-related genes were screened using the STRING database and Cytoscape software. Two downregulated genes, PI3 and MAP2K1, and three upregulated genes, SSTR1, GPER1, and CCR10, were identified by constructing a protein–protein interaction network. Functional enrichment of the differentially expressed genes was analyzed, and the expression of the five key genes in AAA samples was verified using quantitative polymerase chain reaction, which revealed that MAP2K1 was downregulated in AAA, whereas SSTR1, GEPR1, and CCR10 were upregulated; there was no significant difference in PI3 expression. Our study shows that normal vascular and AAA samples can be distinguished via the infiltration of immune cells. Five genes, PI3, MAP2K1, SSTR1, GPER1, and CCR10, may play important roles in the development, diagnosis, and treatment of AAA.

Effect of Activated Protein Kinase C Beta Type Mediated Phosphorylation of Signal Transducer and Activator of Transcription 4 on Proliferation and Phenotypic Transformation of Vascular Smooth Muscle Cells After Vascular Injury Induced by Nano-SiO₂.

The excessive proliferation, endothelial migration, and phenotype transformation of vascular smooth muscle cells (VSMC) lead to increased extracellular matrix secretion, which induces vascular intimal hyperplasia, which is an important restenosis mechanism after vascular injury. In our study, we verified the cytotoxicity of SiO₂ nanoparticles to VSMC. To explore the role of endothelial repairs and molecular mechanisms after vascular injuries, we sequenced the transcriptome of injured vessels in the carotid artery of mice. The results showed the differentially expressed genes in normal vascular tissues, and that vascular tissues were mainly enriched with NF-κB signaling pathways, chemokine signaling pathways and other biological functions, by the leukocyte activation and adhesion of the KEGG pathway in the immune response, and by DNA binding, DNA transcription regulatory region binding, and other molecular functions. Core proteins included PRKCB, STAT4, CCL5, and BCL-2. To verify the roles of these core proteins, RT-qPCR andWestern blot techniques were used to detect their transcription and translation levels, and HE staining was used to detect morphological changes in blood vessels. To further clarify the role of core proteins in VSMC, PRKCB over expression plasmids were constructed, and the RT-qPCR and Western blot techniques were again used to detect the expression of core proteins. The results showed that the levels of transcription and translation, and of PRKCB and STAT4 phosphorylation, increased significantly after vascular injury, and then noticeably decreased three days later- and that CCL5 and Bcl-2 expression trends were consistent with this. HE staining showed that when the vascular endothelium was damaged, smooth muscle cells proliferated significantly, and that the intima thickened three days after vascular injury. After over expression of PRKCB, the expression and activation of STAT4, CCL5, and Bcl-2 significantly increased, α-SMA and Vimentin were down-regulated, OPN was up-regulated, and VSMC activity was enhanced. From these results, it could be concluded that PRKCB is activated by vascular injury, and that over-activation of PRKCB promotes activation of STAT4 and the expression of CCL5 and BCL-2-which in turn leads to enhanced VSMC activity and transformation of its contraction phenotype to the secretion phenotype. We were also able to establish that the cytotoxicity of SiO₂ nanoparticles to VSMC was positively correlated with dose and time.

Fibrovascular Tumor-Like Lesions of the Facial Nerve

Background: Facial nerve tumors (FNTs) are relatively rare benign lesions that arise from any segment of the facial nerve (FN). About half of all patients present with FN dysfunction, mainly long-standing or progressive facial paralysis. Diagnosis of an FNT is usually based on radiological imaging and confirmed by histological study. Most reported cases of FNTs are schwannomas and hemangiomas. Objectives: The aim of this study was to review 4 cases of lesions with clinical, radiological, and surgical findings that suggested an FNT, the pathology revealing a fibrovascular proliferation with no clear signs of a specific tumor. Method: Medical records of patients who had surgery due to an FN lesion were reviewed. Cases with known tumoral lesions were excluded. Four patients with tumor-like lesions were identified. Their imaging studies were re-evaluated. The pathological study included hematoxylin-eosin, Masson’s trichrome, and immunohistochemistry for S100 protein, neurofilaments, CD31, Wilms’ tumor 1 (WT1), and D240. Results: The 4 cases revealed tumor-like fibrovascular lesions that could not be classified as typical pathological entities. All cases had a complete facial palsy preoperatively. Computed tomography and magnetic resonance imaging (MRI) suggested schwannoma or hemangioma. A complete excision was achieved, and a facial reconstruction was performed immediately after interruption. Postoperative FN function was improved in all cases. The histological study showed nervous tracts of normal morphology, with fibrous and vascular tissue interspersed in variable proportions. All cases showed areas of fibrosis with Masson’s stain. In all cases, nervous tissue and Schwann cells tested positively for neurofilaments and S100, respectively. In vascular areas, endothelial cells stained positively for CD31, and negatively for D240 and WT1. Conclusions: Fibrovascular lesions of the FN may mimic primary FNTs, especially schwannomas and hemangiomas. Surgical excision with grafting or nerve transfer is the procedure of choice if a complete facial paralysis is found. This unusual condition should be considered when counseling patients with FN lesions. The lack of hyperintensity on MRI T2-weighted images may suggest the presence of fibrous tissue.

Vascular Microphysiological Systems to Model Diseases.

Human vascular microphysiological systems (MPS) represent promising three-dimensional in vitro models of normal and diseased vascular tissue. These systems build upon advances in tissue engineering, microfluidics, and stem cell differentiation and replicate key functional units of organs and tissues. Vascular models have been developed for the microvasculature as well as medium-size arterioles. Key functions of the vascular system have been reproduced and stem cells offer the potential to model genetic diseases and population variation in genes that may increase individual risk for cardiovascular disease. Such systems can be used to evaluate new therapeutics options.

High-throughput transcriptional profiling combined with angiogenesis antibody array analysis in an orbital venous malformation cohort

Orbital venous malformations (OVMs) are the most common benign orbital vascular disorders in adults and are characterized as enlarging encapsulated vascular neoplasms. These painless lesions grow slowly and become symptomatic with proptosis or visual disturbance. However, the pathogenic mechanism and diagnostic markers of OVMs remain poorly understood. To identify potential pathways involved in OVM formation, a cDNA microarray analysis was conducted with OVM samples and normal vascular tissues. These data were deposited in the National Omics Data Encyclopedia (NODE) database (accession number: OER033009). These pathway expression data were further confirmed by reverse transcription qPCR (RT-qPCR) in an OVM cohort. To explore the diagnostic markers in OVM, an angiogenesis antibody array was analyzed. The altered factors were further validated by enzyme-linked immunosorbent assay (ELISA) in the OVM cohort. Transcriptome screening revealed upregulated autophagy and VEGF pathways and downregulated Hippo, Wnt, hedgehog and vascular smooth muscle contraction signaling pathways in OVM samples. Furthermore, plasma EGF (p < 0.001) and Leptin (p < 0.01) levels were significantly elevated in OVM patients. Here, for the first time, we revealed the transcriptional background and plasma diagnostic markers in OVM, providing a novel understanding of OVM pathogenesis and facilitating the early diagnosis of OVM.

Autophagy and Others Respiratory Diseases.

Besides COPD, pulmonary fibrosis, and asthma, autophagy also participates in the development of many other respiratory diseases. Cystic fibrosis is an innate lung disease. Unlike idiopathic pulmonary fibrosis, cystic fibrosis has unique pathogenesis. Autophagy is an essential biological mechanism for the removal of misfolded proteins and damaged organelles in cells. Abnormal autophagy activity is involved in the pathogenesis of cystic fibrosis. Various studies have demonstrated that abnormalities or impaired autophagy are associated with cardiovascular diseases including pulmonary vascular disease. Autophagy plays a key role in maintaining normal vascular biological functions and vascular cell tissue homeostasis, and also plays an important role in the pathogenesis of various vascular diseases. For example, recent studies have found that autophagy participates in the occurrence and development of pulmonary hypertension. In addition, autophagy plays a central role in both innate and adaptive immune responses in immune cells or other cells with immune function. Thus, autophagy is the important cellular biological mechanism which causes cell fighting against pathogenic microorganisms including viruses, bacteria, and parasites. In this chapter, we discuss the work related to autophagy and other lung diseases.

Nitric Oxide-Releasing Selective Estrogen Receptor Modulators: A Bifunctional Approach to Improve the Therapeutic Index.

When using selective estrogen receptor modulators (SERMs) in cancer therapy, adverse effects such as endothelial dysfunction have to be considered. Estrogens and, consequently, SERMs regulate the synthesis of vasoactive nitric oxide (•NO). We hypothesized that a bifunctional approach combining the antagonistic action of SERMs with a targeted •NO release could diminish vascular side effects. We synthesized a series of NO-releasing SERMs (NO-SERMs) and the corresponding SERMs (after NO release) derived from a triaryl olefin lead. Compounds showed antagonistic activity for ERβ (IC50(ERβ) = 0.2-2.7 μM), but no interaction with ERα. Growth of ERβ-positive breast cancer and melanoma cells was significantly decreased by treatment with SERM 5d. This antiproliferative effect was diminished by the additional release of •NO by the corresponding NO-SERM 4d. Moreover, targeted release of •NO by 4d counteracted the antiproliferative effect of 5d in normal vascular tissue cells. Summarizing, the therapeutic index of SERMs might be improved by this bifunctional approach.

Transcriptional Profiling of Hypoxia-Regulated Non-coding RNAs in Human Primary Endothelial Cells.

Hypoxia occurs in human atherosclerotic lesions and has multiple adverse effects on endothelial cell metabolism. Recently, key roles of long non-coding RNAs (lncRNAs) in the development of atherosclerosis have begun to emerge. In this study, we investigate the lncRNA profiles of human umbilical vein endothelial cells subjected to hypoxia using global run-on sequencing (GRO-Seq). We demonstrate that hypoxia regulates the nascent transcription of ~1800 lncRNAs. Interestingly, we uncover evidence that promoter-associated lncRNAs are more likely to be induced by hypoxia compared to enhancer-associated lncRNAs, which exhibit an equal distribution of up- and downregulated transcripts. We also demonstrate that hypoxia leads to a significant induction in the activity of super-enhancers next to transcription factors and other genes implicated in angiogenesis, cell survival and adhesion, whereas super-enhancers near several negative regulators of angiogenesis were repressed. Despite the majority of lncRNAs exhibiting low detection in RNA-Seq, a subset of lncRNAs were expressed at comparable levels to mRNAs. Among these, MALAT1, HYMAI, LOC730101, KIAA1656, and LOC339803 were found differentially expressed in human atherosclerotic lesions, compared to normal vascular tissue, and may thus serve as potential biomarkers for lesion hypoxia.