Proportion Of Endothelial Cells Research Articles

Tailored endothelialization enabled by engineered endothelial cell vesicles accelerates remodeling of small-diameter vascular grafts

Current gold standard for the replacement of small-diameter blood vessel (ID < 4 mm) is still to utilize the autologous vessels of patients due to the limitations of small-diameter vascular grafts (SDVG) on weak endothelialization, intimal hyperplasia and low patency. Herein, we create the SDVG with the tailored endothelialization by applying the engineered endothelial cell vesicles to camouflaging vascular grafts for the enhancement of vascular remodeling. The engineered endothelial cell vesicles were modified with azide groups (ECVs-N3) through metabolic glycoengineering to precisely link the vascular graft made of PCL-DBCO via click chemistry, and thus fabricating ECVG (ECVs-N3 modified SDVG), which assists inhibition of platelet adhesion and activation, promotion of ECs adhesion and enhancement of anti-inflammation. Furthermore, In vivo single-cell transcriptome analysis revealed that the proportion of ECs in the cell composition of ECVG surpassed that of PCL, and the tailored endothelialization enabled to convert endothelial cells (ECs) into some specific ECs clusters. One of the specific cluster, Endo_C5 cluster, was only detected in ECVG. Consequently, our study integrates the engineered membrane vesicles of ECVs-N3 from native ECs for tailored endothelialization on SDVG by circumventing the limitations of living cells, and paves a new way to construct the alternative endothelialization in vessel remodeling following injury.

Mode of cell death in the penile cavernous tissue of type 1 diabetes mellitus rats.

Diabetes mellitus commonly causes endothelial cell and smooth muscle cell death in penile cavernous tissue. The study sought to study the mode of cell death in the penile cavernous tissue in type 1 diabetic rats. A total of 36 Sprague Dawley rats 10 weeks of age were randomly divided into 2 groups: a normoglycemic group and type 1 diabetic group (intraperitoneal injection of Streptozotocin (STZ), 60mg/kg). We randomly selected 6 rats from each group for tests at the end of 11, 14, and 18weeks of age, respectively. All rats were able to eat and drink freely. The ratio of maximum intracavernous pressure to mean arterial pressure, concentration of serum testosterone, level of nitric oxide in the penile cavernosum, and expression of active caspase-1 (pyroptosis) and active caspase-3 (apoptosis) were determined. At the end of weeks 4 and 8 of type 1 diabetes, the proportions of endothelial cells and smooth muscle cells undergoing apoptosis and pyroptosis in penile cavernous tissue are different. The ratio of maximum intracavernous pressure to mean arterial pressure and nitric oxide levels were significantly lower in the 4- and 8-week diabetic groups than in the normoglycemic group (P < .01). Penile endothelial cell pyroptosis (5.67 ± 0.81%), smooth muscle cell apoptosis (23.72 ± 0.48%), total cell pyroptosis (9.67 ± 0.73%), and total apoptosis (10.52 ± 1.45%) were significantly greater in the 4-week diabetic group than in the normoglycemic group (P < .01). The proportion of endothelial cell pyroptosis (24.4 ± 3.69%), endothelial cell apoptosis (22.13 ± 2.43%), total cell pyroptosis (14.75 ± 0.93%), and total apoptosis (14.82 ± 1.08%) in the penile tissues of the 8-week diabetic group were significantly greater than those in the normoglycemic group (P < .01).The 8-week survival proportions of diabetic endothelial cells (38.86 ± 8.85%) and smooth muscle cells (44.46 ± 2.94%) was significantly lower than the 4-week survival proportions of endothelial cells (93.17 ± 8.07%) and smooth muscle cells (75.12 ± 4.76%) (P < .05). Inhibition of cell death by different methods at different stages may be the key to the treatment of type 1 diabetes-induced erectile dysfunction. The effect of type 1 diabetes on other types of cell death in penile cavernous tissue needs further study. The mode of death of endothelial cells in the cavernous tissue of the penis in the early stage in diabetic rats is dominated by pyroptosis, and the death of smooth muscle cells is dominated by apoptosis. Endothelial cell and smooth muscle cell death are not consistent at different stages of diabetes progression.

Ductal carcinoma in situ: Molecular and cellular basis of malignancy.

e12582 Background: The goal of our study is to improve the diagnosis and treatment of DCIS through the identification of the factors driving its invasion and metastasis. Methods: We utilized the Mouse INtraDuctal (MIND) model to investigate the mechanism(s) by which some DCIS become invasive while others remain indolent. This model closely replicates the human condition by injecting non-invasive epithelial cells from patients into mouse mammary ducts to form in situ lesions. In some cases, these cells will display invasive behavior by overcoming the natural barriers of myoepithelium. To compare these two processes, we performed single-cell (sc) RNA-sequencing analysis of 60,000 cells from the pre-transplant time-point representing 17 DCIS (11 became invasive, 6 remained non-invasive). We also performed scATAC/RNA sequencing of a DCIS with associated IDC. Results: Our analysis revealed that the proportion of endothelial cells was higher in cases that became invasive, while the proportion of plasma and T cells was higher in those that remained non-invasive (t-test; P&lt;0.05). This finding suggested that invasive cells reprogrammed the immune microenvironment to exclude immune cells as a means of evading immune surveillance. Additionally, we performed cell-cell interaction (CCI) analysis by CellPhoneDB. This analysis revealed highly ranked CCIs in invasive cases, including luminal cell interactions with pericytes via Wnt5A-SFRP1. On the other hand, highly ranked CCIs in non-invasive cases included HR-positive luminal cell interactions with T cells via CD226-Nectin-2 and CD96-Nectin1. Notably, CD226 and CD96 are T cell receptors that enhance CD8+ T cell activity. These results support the potential role of T-cell interactions with DCIS luminal cells in preventing their invasive transition. We conducted additional analysis of sc-RNA-sequencing data using a stem cell-based gene signature algorithm called CytoTRACE. Our analysis revealed that invasive cells were enriched in cell clusters with significantly higher stem cell scores than non-invasive cells. In another analysis of a patient DCIS with associated IDC using sc-ATAC and RNA sequencing, we discovered that the open chromatin regions in the stem cell cluster were enriched for FOXA1 binding motifs. This cluster also had the highest expression of two pioneer factors (FOXA1 and GRHL2), NEAT1 (paraspeckle protein), ERBB2, 3,4, and ANKRD30A (cell surface protein). Our findings suggest that the pioneer factors FOXA1 and GRHL2 play a crucial role as transcriptional drivers of stemness. Additionally, the ANKRD30A could be used to isolate stem cell clusters to validate their self-renewal potential. Conclusions: DCIS progression is facilitated by interactions between stromal, immune, and epithelial cells, which lead to epigenetic and transcriptional changes. This results in the formation of stem-like cells that are characterized by their plasticity and invasive capacity.

Single-cell RNA sequencing reveals hub genes of myocardial infarction-associated endothelial cells

BackgroundMyocardial infarction (MI) is a cardiovascular disease that seriously threatens human health. Dysangiogenesis of endothelial cells (ECs) primarily inhibits recovery from MI, but the specific mechanism remains to be further elucidated.MethodsIn this study, the single-cell RNA-sequencing data from both MI and Sham mice were analyzed by the Seurat Package (3.2.2). The number of ECs in MI and Sham groups were compared by PCA and tSNE algorithm. FindMarkers function of Seurat was used to analyze the DEGs between the MI and Sham groups. Then, the ECs was further clustered into 8 sub-clusters for trajectory analysis. The BEAM was used to analyze the branch point 3 and cluster the results. In addition, the DEGs in the microarray data set of MI and Sham mice were cross-linked, and the cross-linked genes were used to construct PPI networks. The key genes with the highest degree were identified and analyzed for functional enrichment. Finally, this study cultured human umbilical vein endothelial cells (HUVECs), established hypoxia models, and interfered with hub gene expression in cells. The impact of hub genes on the migration and tube formation of hypoxic-induced HUVECs were verified by Wound healing assays and tubule formation experiments.ResultsThe number and proportion of ECs in the MI group were significantly lower than those in the Sham group. Meantime, 225 DEGs were found in ECs between the MI and Sham groups. Through trajectory analysis, EC4 was found to play an important role in MI. Then, by using BEAM to analyze the branch point 3, and clustering the results, a total of 495 genes were found to be highly expressed in cell Fate2 (mainly EC4). In addition, a total of 194 DEGs were identified in Micro array dataset containing both MI and Sham mice. The hub genes (Timp1 and Fn1) with the highest degree were identified. Inhibiting Timp1 and Fn1 expression promoted the migration and tube formation of HUVECs.ConclusionsOur data highlighted the non-linear dynamics of ECs in MI, and provided a foothold for analyzing cardiac homeostasis and pro-angiogenesis in MI.

Single Cell RNASeq Analysis of AD and Control Post Mortem Brain Samples

Alzheimer’s disease (AD) is a devastative late life disease, molecularly characterized by biochemical lesions of amyloid β plaques and Tau tangles. the 2nd most frequent one worldwide with no early detection methods/cure. I have applied single cell sequencing and analysed post mortem brain samples (using a Chromium machine) from 2 AD and 2 control frontal cortex samples. All the samples had ethics approval, and the RNA was tested for quality. The samples were obtained from the Edenborough MRC brain bank. Very few genes have been linked to far to AD (mainly through GWAS studies [1]). Overall, I quantified about 24,000 nuclei. Using statistical, bioinformatics and computational analyses I have detected significant differential expression changes in astrocyte, neuronal and microglial marker genes. I also conducted network analyses. The percentage of reads passing filter was slightly low (68%). Overall output was a bit lower than optimal. There was still over 1.6Bn clusters generated though, which is within the specification for an S1 flow-cell, and base call quality (quality control) was excellent: 97% &gt;Q30. The read counts mapping to genome occupancy rate was also fairy low (63-81%). The number of barcodes is as follows: sample 1 (AD, Braak I-II) - 6,223 sample 2 (control) 6,262 sample 3 (AD Braak I-II) 5,332 sample 4 (AD, Braak I-II) 5,332 as well. The data is available for download under the GEO (accession number GSE175814). to assess global and cell-type specific gene expression impacts in Alzheimer’s versus control brain samples, we performed a holistic comparison of matching cell populations in AD and controls with the software cell Harmony. To identify possible gene regulatory network underlying transcriptional regulation in these distinct populations, we further explored prior evidenced transcription factor targets relationships from cell Harmony. Importantly, we observe both common and cell-type specific core transcription factors regulators, with both enriched downstream targets and regulated transcription factors. Upregulated networks in Astrocytes were associated with broad regulation by HIF1A, SOX2, NRF1, and RB1. the proportion of endothelial cells was higher in the Alzheimer’s disease samples than the neurological control samples. results reveal that the cell type-specific transcriptomic changes in Alzheimer’s disease are associated with 4 molecular pathways: angiogenesis in endothelial cells, immune response in endothelial cells and microglia, myelination in oligodendrocytes, and synaptic signalling in astrocytes and neurons. I also compared the results with microarray data from large cohort studies that examined samples from the prefrontal cortex (Alzheimer’s disease: n = 310; Neurological controls: n = 157) or temporal cortex (Alzheimer’s disease: n = 106; Neurological controls: n = 135). Among the DEGs identified in our snRNA-seq analysis, 1,113 and 764 genes were significantly differentially expressed in the microarray data from the prefrontal cortex and temporal cortex, respectively. subcluster analysis of microglia identified 13 subpopulations; VGF signalling was also found as changed. In the future, imaging of post mortem Alzheimer’s patients may verify brain cellular morphology/cell volume of vascular changes in the Alzheimer’s compared to control samples. My study findings [2] may open new venues to our understanding of the disease underlying molecular processes and cellular signaling. Regarding the glial cell changes, our main conclusion is that the glial cells may be reactive in the disease and not causal.

Single-cell RNA transcriptome landscape of murine liver following systemic administration of mesoporous silica nanoparticles

Due to the unique physicochemical properties, mesoporous silica nanoparticles (MONs) have been widely utilized in biomedical fields for drug delivery, gene therapy, disease diagnosis and imaging. With the extensive applications and large-scale production of MONs, the potential effects of MONs on human health are gaining increased attention. To better understand the cellular and molecular mechanisms underlying the effects of MONs on the mouse liver, we profiled the transcriptome of 63,783 single cells from mouse livers following weekly intravenous administration of MONs for 2 weeks. The results showed that the proportion of endothelial cells and CD4+ T cells was increased, whereas that of Kupffer cells was decreased, in a dose-dependent manner after MONs treatment in the mouse liver. We also observed that the proportion of inflammation-related Kupffer cell subtype and wound healing-related hepatocyte subtype were elevated, but the number of hepatocytes with detoxification characteristics was reduced after MONs treatment. The cell-cell communication network revealed that there was more crosstalk between cholangiocytes and Kupffer cells, liver capsular macrophages, hepatic stellate cells, and endothelial cells following MONs treatment. Furthermore, we identified key ligand-receptor pairs between crucial subtypes after MONs treatment that are known to promote liver fibrosis. Collectively, our study explored the effects of MONs on mouse liver at a single-cell level and provides comprehensive information on the potential hepatotoxicity of MONs.

Premature differentiation of nephron progenitor cell and dysregulation of gene pathways critical to kidney development in a model of preterm birth

Preterm birth is a leading cause of neonatal morbidity. Survivors have a greater risk for kidney dysfunction and hypertension. Little is known about the molecular changes that occur in the kidney of individuals born preterm. Here, we demonstrate that mice delivered two days prior to full term gestation undergo premature cessation of nephrogenesis, resulting in a lower glomerular density. Kidneys from preterm and term groups exhibited differences in gene expression profiles at 20- and 27-days post-conception, including significant differences in the expression of fat-soluble vitamin-related genes. Kidneys of the preterm mice exhibited decreased proportions of endothelial cells and a lower expression of genes promoting angiogenesis compared to the term group. Kidneys from the preterm mice also had altered nephron progenitor subpopulations, early Six2 depletion, and altered Jag1 expression in the nephrogenic zone, consistent with premature differentiation of nephron progenitor cells. In conclusion, preterm birth alone was sufficient to shorten the duration of nephrogenesis and cause premature differentiation of nephron progenitor cells. These candidate genes and pathways may provide targets to improve kidney health in preterm infants.

Isolation and characterization of the human ovarian cell population for transplantation into an artificial ovary.

To support survival and growth of follicles, the transplantable artificial ovary should mimic the original organ, offering a physical (3D matrix) and biological support (cells). In order to replicate the ovarian cell populations, the aim of this study is to assess the proportions of stromal and endothelial cells in the ovarian cortex. To this end, ovarian biopsies were obtained from six women (mean age: 49 years). The epithelial layer and medulla were carefully removed. The cortex was finely minced and enzymatically digested and the isolated cells were fixed. For cell characterization, immunostaining for CD31 (for endothelial cells) and inhibin-α (for granulosa cells) was performed. Positive cells in each staining were counted and the proportion of the different cell populations was estimated from the total number of isolated cells. Since there is no specific marker for ovarian stromal cells, we estimated the proportion of these cells by performing a vimentin immunostaining and subtracting the proportions of CD31- and inhibin-α-positive cells. Immunostaining showed that 84% of isolated cells were vimentin-positive. From this pool, 3% were endothelial cells and 1% granulosa cells. Consequently, the population of ovarian stromal cells was 80%. In conclusion, our findings show that stromal cells represent the larger population of cells in the human ovarian cortex. While this ensures follicle survival and development in a normal ovary, we believe that the low proportion of endothelial cells could have a negative impact on the angiogenesis in the artificial ovary after the first days of transplantation.

Abstract 182: Cancer stem cells and tumor angiogenesis in serous adenocarcinoma of ovary

Abstract The traditional view of tumour vascularization is that tumours acquire blood supply from the neighbouring normal stroma. However, recently the origin of tumour endothelial cells or pericytes in part has been shown to be derived from cancer stem cells (CSCs) in glioblastoma. In high grade serous ovarian cancer (HGSOC), the origin of endothelial cells is not known. Our objective was to determine if components of a tumour blood vessel and lymphatic vessel are derived from CSCs in ovarian cancer. Using spheroids as an in vitro model, we have evaluated the role of CSCs in primary malignant cells (PMCs) from patients with serous adenocarcinoma of ovary cultured under specific conditions. The expression of endothelial, pericyte and lymphatic endothelial markers was evaluated by flow cytometry. In addition, functional assays were performed to assess the endothelial phenotype. Further, the ability of CSCs to express endothelial markers under appropriate growth conditions was also evaluated with Bevacizumab which antagonize VEGF. PMCs grown in endothelial growth medium (EGM) showed significantly higher expression of CD105 (n=32, p = 0.002) and CLEC14A (n=10, p = 0.012) and co-expression of CD105/CLEC14A (n=10, p= 0.012) than that of spheroids. Primary malignant cells when grown in pericyte and lymphatic endothelial specific conditions, showed significantly higher expression of desmin (n=10, p=0.03), Smooth muscle actin (SMA) (n=10, p=0.017) and VEGFR3 (n=10, p=0.028) than that of the spheroids. When the PMCs were grown as spheroids in endothelial conditions in the presence or absence of Bevacizumab (1 μg/μl), there was a reduction in the co-expression of CD105 and CLEC14A (P=0.04). The cells grown in endothelial conditions showed formation of tubes, uptake of Dil-ac-LDL and expressed eNOS, confirming their endothelial phenotype. GFP transduced spheroids from PMCs formed tumours in mice and the blood vessels in the tumour co-expressed CD31, SMA, VEGFR3 and GFP, suggesting that these cells are derived from CSCs. These results prove that a proportion of endothelial cells, pericytes and lymphatic endothelial cells are derived from CSCs in serous ovarian carcinoma and the VEGF pathway has a key role. This property of CSCs to contribute to tumour angiogenesis can be inhibited. Citation Format: S Krishnapriya, C Sidhanth, P Manasa, S Sneha, S Bindhya, R P. Nagare, T S. Ganesan. Cancer stem cells and tumor angiogenesis in serous adenocarcinoma of ovary [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 182.

Method for in vitro differentiation of bone marrow mesenchymal stem cells into endothelial progenitor cells and vascular endothelial cells

Vascular development is a regulated process and is dependent on the participation and differentiation of many cell types including the proliferation and migration of vascular endothelial cells and differentiation of endothelial progenitor cells (EPCs) to mesodermal precursor cells. Thus, reconstitution of this process in vitro necessitates providing ambient conditions for generating and culturing EPCs in vitro and differentiating them to vascular endothelial cells. In the present study, we developed methods to differentiate bone marrow mesenchymal stem cells (MSC) into EPCs and to vascular endothelial cells. Bone marrow MSC from canines and human sources were differentiated in vitro in to EPCs. These EPCs were able to express a variety of endothelial markers following 7 days in culture. Further culturing led to the appearance of an increased number and proportion of endothelial cells. These cells were stable even after 30 generations in culture. There was a gradual loss of CD31 and increased expression of factor VIII, VEGFR and CD133. VEGF being highly angiogenic, helps in the vascular development. These results provide the basis for the possible development of vasculature in vitro conditions for biomedical applications and in vivo for organ/tissue reconstruction therapies.

Abstract 3330: Tumor angiogenesis and cancer stem cells in serous adenocarcinoma of the ovary

Abstract Angiogenesis is required for tumor growth and metastasis. The conventional view of tumour angiogenesis is that tumours get their blood supply from the neighbouring normal stroma. However, recently the origin of tumour endothelial cells or pericytes in part has been shown to be derived from cancer stem cells (CSC) in glioma. The spread of ovarian cancer (SOC) is different and the origin of endothelial cells in the tumor is not known. Using spheroids as an in vitro model (which is enriched for CSC), we have evaluated the role of CSC in primary malignant cells (PMCs) from patients with serous adenocarcinoma of ovary (n = 30) cultured under endothelial conditions. The expression of endothelial markers (CD31, CD105, and CLEC14a) was evaluated by flow cytometry. Further, the ability of CSC to express endothelial markers under appropriate growth conditions was also evaluated with Bevacizumab (Avastin) or Cediranib which antagonize VEGF or its receptor (VEGFR2) respectively. In addition, functional assays such as uptake of Dil labelled acetylated low density lipoprotein (Dil-ac-LDL) and expression of endothelial nitric oxide synthase (e-NOS) was performed to assess the endothelial phenotype. The localization of tumour blood vessels and CSC in primary ovarian tumors was examined by double immunohistochemistry of CD31/CD105/CLEC14a and ALDH1a1. Primary malignant cells (n = 30) grown in endothelial growth medium (EGM) showed significantly higher expression of CD105 (mean 20.8%, p = 0.001) and CLEC14a (mean 5.3%, p = 0.012) and a co-expression of CD105/CLEC14a (mean 1.6%, p = 0.012) than that of control (mean, 12.5%, 2.5% and 0.95% respectively). The co-expression of ALDH1a1 with endothelial markers, CD31, (mean, 1.58%), CD105, (mean 0.7%), CLEC14a (mean 0.44%) in primary malignant cells (n = 5), denovo, suggest that there is a small proportion of cells which are in transit to form endothelial cells. When the primary malignant cells were grown as spheroids in endothelial conditions in the presence or absence of Avastin (1 μg/μl), there was reduction in the expression of CD105 (mean, 12.5% (EGM) and 7.9% (Avastin), P = 0.056) and CLEC14a (mean. 5.5% (EGM) and 1.5% (Avastin), P = 0.04). However, when the spheroids were cultured in presence of cediranib (10 nM), the expression of CD105 was not reduced (Mean, 2.53% (EGM) and 4% (cediranib). The cells grown in endothelial conditions showed uptake of Dil-ac-LDL (n = 3) and expressed e-NOS (n = 3), confirming their endothelial phenotype. The double immunostaining with ALDH1a1 and CD31/CD105 demonstrated that the blood vessels were in proximity to ALDH1A1+ cells in primary serous adenocarcinoma of the ovary (n = 5). These results suggest that a proportion of endothelial cells (probably 20%) could be derived from CSC in serous ovarian carcinoma and the VEGF pathway has an important role. This property of CSC to contribute to tumour angiogenesis can be inhibited. Citation Format: Syama Krishna Priya, Rohit P. Nagare, V S. Sneha, C Sidhanth, S Bindhya, P Manasa, Trivadi S. Ganesan. Tumor angiogenesis and cancer stem cells in serous adenocarcinoma of the ovary. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3330.

A single fusion signal for t(14;18)(q32;q21) translocation is present in both the follicular lymphoma and local endothelial cells

Herein we reported a case of follicular lymphoma with 50.26% clonal malignant lymphocytes and 50% tumor cells positive for the immunoglobulin heavy chain gene and B-cell lymphoma 2 gene (IGH-BCL2). To determine whether endothelial cells (ECs) within the tumor share the feature of advanced malignancy, we isolated and purified the ECs from the tumor by using the immunomagnetic beads conjugated with a monoclonal antibody against CD34, a surface marker of ECs. Thereafter, we identified ECs according to their morphology and found that ECs presented consistently flat and elongated appearance with a lot of Weibel-Palade bodies in the cytoplasm. Results of flow cytometry confirmed that ECs isolated from the follicular lymphoma expressed high level of both vWF and CD34 and the purity of the ECs fraction was more than 90%. Additionally, we used FISH to check chromosomal aberration in the purified ECs and found that some of the ECs had only one fusion signal for the green IGH probe and the red BCL2 probe in contrast to typical t(14;18)(q32;q21) translocation with two fusion signals. This phenomenon was also observed in the tumor cells. It might be a different breakpoint of IGH in this case, which induced the loss of the fusion signal, indicating t(14;18)(q32;q21) translocation. The positive cells accounted for 18% of the isolated ECs from the tumor, indicating that a proportion of ECs from follicular lymphoma had the same chromosome aberration as the neoplastic cells.

Human Placenta Is a Potent Hematopoietic Niche Containing Hematopoietic Stem and Progenitor Cells throughout Development

Hematopoietic stem cells (HSCs) are responsible for the life-long production of the blood system and are pivotal cells in hematologic transplantation therapies. During mouse and human development, the first HSCs are produced in the aorta-gonad-mesonephros region. Subsequent to this emergence, HSCs are found in other anatomical sites of the mouse conceptus. While the mouse placenta contains abundant HSCs at midgestation, little is known concerning whether HSCs or hematopoietic progenitors are present and supported in the human placenta during development. In this study we show, over a range of developmental times including term, that the human placenta contains hematopoietic progenitors and HSCs. Moreover, stromal cell lines generated from human placenta at several developmental time points are pericyte-like cells and support human hematopoiesis. Immunostaining of placenta sections during development localizes hematopoietic cells in close contact with pericytes/perivascular cells. Thus, the human placenta is a potent hematopoietic niche throughout development.

FGFR-1 is required by epicardium-derived cells for myocardial invasion and correct coronary vascular lineage differentiation

Critical steps in coronary vascular formation include the epithelial–mesenchyme transition (EMT) that epicardial cells undergo to become sub-epicardial; the invasion of the myocardium; and the differentiation of coronary lineages. However, the factors controlling these processes are not completely understood. Epicardial and coronary vascular precursors migrate to the avascular heart tube during embryogenesis via the proepicardium (PE). Here, we show that in the quail embryo fibroblast growth factor receptor (FGFR)-1 is expressed in a spatially and temporally restricted manner in the PE and epicardium-derived cells, including vascular endothelial precursors, and is up-regulated in epicardial cells after EMT. We used replication-defective retroviral vectors to over-express or knock-down FGFR-1 in the PE. FGFR-1 over-expression resulted in increased epicardial EMT. Knock-down of FGFR-1, however, did not inhibit epicardial EMT but greatly compromised the ability of PE progeny to invade the myocardium. The latter could, however, contribute to endothelia and smooth muscle of sub-epicardial vessels. Correct FGFR-1 levels were also important for correct coronary lineage differentiation with, at E12, an increase in the proportion of endothelial cells amongst FGFR-1 over-expressing PE progeny and a decrease in the proportion of smooth muscle cells in antisense FGFR-1 virus-infected PE progeny. Finally, in a heart explant system, constitutive activation of FGFR-1 signaling in epicardial cells resulted in increased delamination from the epicardium, invasion of the sub-epicardium, and invasion of the myocardium. These data reveal novel roles for FGFR-1 signaling in epicardial biology and coronary vascular lineage differentiation, and point to potential new therapeutic avenues.

Rates of Cycling Cells in Cryopreserved Valvular Homograft: A Preliminary Study

Some investigators claim that the viability of cryopreserved human valvular homograft is necessary for the duration of implanted homograft. In this preliminary study, the percentage of cycling cells in cryopreserved valvular homografts was evaluated with the use of monoclonal Ki-67 antibody. Three human aortic valves were harvested from multiorgan donors and cryopreserved. Sections of 5 microm in thickness were stained with monoclonal Ki-67 antibody. The proportion of endothelial cells with Ki-67 positive nuclei was 1.80 +/- 0.20%. No differences in distribution were observed from basal to marginal sites. Few fibroblasts showed Ki-67-immunopositivity (0.10 +/- 0.06%) while the Ki-67 immunostaining was 0.80 +/- 0.20% in myocytes. Our preliminary study shows that cryopreserved valvular homograft cells are not only viable but they also have the potential to replicate. These data can lead to the hypothesis that valvular cells could actively replicate even after implantation, permitting cellular renewal and regeneration of extracellular matrix's components.

Troglitazone reverses the inhibition of nitric oxide production by high glucose in cultured bovine retinal pericytes

In the retinal microcirculation, there is a basal release of nitric oxide (NO) which maintains the retinal blood flow. The proportions of endothelial cells and pericytes in the retinal capillaries are almost equal, so pericytes appear to play a important role in the regulation of microcirculatory hemodynamics in the retina. It has been suggested that the pathogenesis of early diabetic retinopathy may involve a reduced bioavailability or diminished production of NO. In this study, we investigated the role of troglitazone, a potent agonist of peroxisome proliferator activated receptor-gamma (PPARγ) used for the treatment of diabetes, on the NO release and the effect of exposure to high glucose on the production of NO in cultured bovine retinal pericytes. Troglitazone significantly increased NO production and iNOS expression after 24 hr in a dose-and PPARγ-dependent manner. Elevation of d-glucose, but not l-glucose, from 5.5 to 30 m m for 24 hr decreased NO production, but co-treatment with troglitazone reversed high glucose-induced inhibition of NO production as well as iNOS expression. In conclusion, high glucose inhibits iNOS expression and subsequently NO synthesis in cultured bovine retinal pericytes, and troglitazone restores the NO production.

Dual-Label Immunohistochemical Study of Interleukin-4-and Interferon-γ-Expressing Cells within the Pancreas of the NOD Mouse During Disease Acceleration with Cyclophosphamide

Beta cell destruction has been shown to occur when rodent or human islets are exposed in vitro to inflammatory cytokines, such as interleukin-1β″ (IL-1β), tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). Other cytokines such as interleukin-4 (IL-4) or inter-leukin-10 (IL-10), when given to NOD mice, prevent insulin-dependent diabetes mellitus (IDDM). In this study, we have employed immunofluorescence histochemistry to study the expression of IFN-γ and IL-4 in the pancreas of female NOD mice at various time-points (days 0, 4, 7, 11 and at onset of diabetes) following disease acceleration with cyclophosphamide (Cy). Dual-label confocal and light microscopy were employed to determine the precise cellular sources of the two cytokines. IL-4 immunolabelling was observed in a few immune cells at days 0, 4, and 7 within the pancreatic islets but in larger numbers at day 11 and at onset of diabetes. The cytokine was co-localized predominantly in CD4 cells, while only a small minority of CD8 cells and macrophages also expressed IL-4. At days 0, 4, 7 and 11, weak to moderate immunolabelling for IL-4 was also observed in beta cells. In contrast, immunolabelling for IFN-γ within the islets was not observed until day 11 and this labelling persisted at onset of diabetes. It was immunolocalized in macrophages and to a lesser extent in CD4 cells. Only a few CD8 cells were immunopositive for IFN-γ. At day 11, a proportion of beta cells showed weak immunolabelling for IFN-γ. During the study period, immunolabelling for IFN-γ was also observed in a proportion of endothelial cells located in the intra-islet and exocrine regions of Cy and diluent-treated mice. From day 11 onwards, both the cytokines were observed in some of the peri-vascular regions. Our results demonstrate that during Cy-induced diabetes, there is increasing expression of both IL-4 and IFN-γ in specific immune cells within the inflamed islets in the late prediabetic stage and at onset of diabetes. Further studies are required to correlate our protein immunohistochemical findings with in situ cytokine gene expression and to determine whether there is a clear Thl cytokine protein bias at clinical onset of diabetes and immediately preceding it

Early captopril treatment inhibits DNA synthesis in endothelial cells and normalization of maximal coronary flow in infarcted rat hearts.

Cardiac remodeling due to myocardial infarction (MI) includes myocyte hypertrophy, collagen deposition, a rise in DNA synthesis, and normalization of initially diminished maximal coronary bloodflow. Previously, we demonstrated that early captopril treatment can prevent the rise in total DNA synthesis, collagen deposition and hypertrophy. In the present experiments, we investigated the effects of captopril or perindoprilat treatment on cardiac endothelial cell proliferation and maximal coronary flow. MI was induced by ligation of the left coronary artery in Wistar rats. Sham-operated and infarcted rats were treated with captopril (12 mg/kg.d s.c.) from either day 0-21 (early) or day 21-35 (late) after surgery. In isolated retrogradely perfused rat hearts, maximal coronary flow was determined following maximal dilatation with nitroprusside and adenosine (1 mM each). In separate groups, sections of hearts of sham-operated and MI rats treated with BrdU (day 7-14) and either captopril or perindoprilat (1 mg/kg.d s.c.; day 0-14) were double stained with a monoclonal anti-BrdU antibody and the lectin GSI. The total fraction of DNA synthesizing cells and its proportion of endothelial cells was determined. Maximal coronary flow was completely normalized in MI hearts within three weeks after surgery. Early captopril, but not late captopril, inhibited the normalization of maximal coronary flow in MI hearts (Early: sham, 27.4 +/- 1.0; MI, 21.2 +/- 1.4 ml/min; P < 0.05; mean +/- SEM) without affecting the hypertrophic response. The total fraction of DNA synthesizing cells was significantly increased in MI hearts (sham: 7.6 +/- 1.9; MI: 14.9 +/- 2.2%). The proportion of endothelial cells, however, was comparable in sham-operated and infarcted hearts (sham: 30 +/- 3; MI: 33 +/- 3%). Both early captopril and perindoprilat treatment inhibited total DNA synthesis in MI hearts. Only in captopril pre-treated hearts, this inhibition was associated with a disproportionate inhibition of the endothelial cell proliferation (10.3 +/- 2.0%). Early captopril treatment inhibits endothelial cell proliferation and coronary vessel growth following MI, which seems to be partly due to inhibition of the renin angiotensin system.

Capillary growth in overloaded, hypertrophic adult rat skeletal muscle: an ultrastructural study.

We examined the early stages of angiogenesis in overloaded m. extensor digitorum longus following extirpation of the agonist m. tibialis anterior. Capillary-to-fibre ratio increased after 1 week (1.54+/-0.02) vs. control (1.38+/0.06; P < 0.01) and resulted in a greater tortuosity of the capillary bed at 2 weeks, indicating the presence of lateral sprouts or anastomoses. Capillary endothelial cells (ECs) showed ultrastructural signs of activation, were thickened, and had irregular luminal and abluminal surfaces. The proportion of ECs with abluminal processes increased after overload (13.5+/-0.6% vs. 2.0+/-1.5%, 1 week vs. contralateral, P < 0.01; 12.5+/-2.6% vs. 3.5+/-0.6%, 2 weeks vs. contralateral, P < 0.01), whereas there was no significant change in proportion of luminal processes. Abluminal processes occurred in approximately 13% of capillaries in overloaded muscles (P < 0.01 v. control and contralateral), and most were associated with focal breakage of the basement membrane (BM). Small sprouts (<3 microm in diameter) comprised of one or two ECs sometimes lacked a lumen, and others had a slitlike or vacuolelike lumen between adjacent ECs or vacuolelike lumen formed by fusion of vesicles within a single EC. Endothelial mitosis was occasionally seen in nonsprouting capillaries with intact BM, increasing the average number of ECs per capillary from approximately 1.7 in control muscles to 2.1 after 1 week of overload (P < 0.05) when bromodeoxyuridine incorporation was also higher (P < 0.001). We conclude that muscle overload induces capillary growth by sprouting of existing capillaries, probably due to mechanical stretch acting from the abluminal side of the vessels.

Cytokine mRNA expression of bone marrow stromal cells from patients with aplastic anaemia and myelodysplastic syndrome.

We studied mRNA expression of the cytokine granulocyte-colony stimulating factor (G-CSF), interleukin-1 beta (IL-1 beta), IL-6, IL-8 and stem cell factor of stromal cells derived from bone marrows of nine normal volunteers, eight patients with aplastic anaemia (AA) and seven patients with myelodysplastic syndrome (MDS). The proportion of endothelial cells, macrophages, fibroblast-like cells and adipocytes in stromal cells showed no differences between normal volunteers and the patients. Levels of cytokine mRNA expression were determined by reverse transcription-polymerase chain reaction. Spontaneous expression occurred and this was augmented by LPS stimulation in cells of all the normal volunteers and in most patients. When stimulated by LPS, the mean G-CSF and IL-1 beta mRNA expressions in patients with AA were significantly higher than normal volunteers, but there was one patient showing lower IL-1 beta, IL-6 and IL-8 expression with no response to LPS. LPS-induced IL-6 and IL-8 expression of two patients with MDS were significantly higher than normal. The spontaneous and LPS-induced protein concentration of G-CSF, IL-6 and IL-8 in culture supernatants from 15, 10 and four patients, correlated well with the mRNA expression. The correlation coefficients were 0 x 92, 0 x 78 and 0 x 91, respectively. In conclusion, there were a few patients whose aetiology appeared to be reduction of stromal cytokine expression in AA, but most patients with AA and MDS expressed normal or high levels of cytokine mRNA.