A robust and efficient microvascular isolation method for multimodal characterization of the mouse brain vasculature
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA884993 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Studying disease related changes in the brain vasculature is pivotal due to its crucial role in supplying oxygen and nutrients, and removing waste. To study the vascular cells in detail, improved techniques for isolation of the vascular components are warranted. In this study, 22,515 cells were isolated using CD31 coupled magnetic beads and analyzed using single cell RNA sequencing. Their transcriptomes separated into 23 clusters corresponding to all known vascular and perivascular cell types. Western blot analysis using markers for these cell types confirmed their presence in the isolate and suggests that the protocol is suitable also for proteomic analysis. Finally, we adapted the protocol to accommodate primary culture. In conclusion, this method can successfully isolate murine brain microvasculature independent of cell sorting, alleviating the need for reporter mouse lines. The protocol is suitable for several testing modalities, including single-cell analyses, WB, and primary cell culture.
2022-10-01 | GSE214326 | GEO
Project description:Studying disease-related changes in the brain vasculature is warranted due to its crucial role in supplying oxygen and nutrients and removing waste and due to the anticipated vascular dysfunction in brain diseases. To this end, we have developed a protocol for fast and simple isolation of brain vascular fragments without the use of transgenic reporters. We used it to isolate and analyze 22,515 cells by single-cell RNA sequencing. The cells distributed into 23 distinct clusters corresponding to all known vascular and perivascular cell types in the brain. Western blot analysis also suggested that the protocol is suitable for proteomic analysis. We further adapted it for the establishment of primary cell cultures. The protocol generated highly reproducible results. In conclusion, we have developed a simple and robust brain vascular isolation protocol suitable for different experimental modalities, such as single-cell analyses, western blotting, and primary cell culture.
| S-EPMC10088242 | biostudies-literature
Project description:Tumor vasculature are structurally chaotic and functionally inefficient. Restoring aberrant tumor blood vessels, or “normalize” the tumor vasculature can alleviate hypoxia and enhance intra-tumoral drug delivery. However, identifying tumor vascular normalizing drugs are currently hampered by an absence of efficient screening platform. We aimed to develop a robust method to visualize, digitalize and evaluate the structural and functional changes of tumor vasculature in batch: zebrafish functional xenograft vasculature platform (zFXVP). As proof of principle, we applied zFXVP to a small compound library which has been implicated in affecting the morphology of tumor vasculature. zFXVP identified PF-502 as a durable tumor vascular normalization agent. Further molecular analysis using RNA-Seq, pharmaceutical inhibition and gene knockout indicate PF-502 can induce endothelial cell cycle arrest and simplify redundant tumor vasculature by blocking PI3K/mTOR signaling and stabilize the vessel lumen and stimulate the blood function by activating Notch1 signaling with mediating S3 cleavage. Last, in mouse tumor xenograft model, PF-502 reproduced a potent vascular normalizing effect at a sub MTD (maximum tolerated dose) and showed its ability to improve intra-tumoral drug delivery and synergize chemotherapy, which confirms the reliability of zFXVP and implicates the potential application of PF-502 in clinical practice as an adjuvant vascular normalization drug.
2023-09-13 | GSE224106 | GEO
Project description:Pulmonary hypertension (PH) is a life-threatening disease, characterized by excessive pulmonary vascular remodeling, leading to elevated pulmonary arterial pressure and right heart hypertrophy. PH is caused, among other factors, by chronic hypoxia, leading to hyper-proliferation of pulmonary arterial smooth muscle cells (PASMC) and apoptosis-resistant pulmonary microvascular endothelial cells (PMVEC). Upon re-exposure to normoxia, chronic hypoxia-induced PH in mice is reversible. In this study, we aim to identify novel candidate genes involved in pulmonary vascular remodeling specifically in the pulmonary vasculature.
2022-09-01 | GSE183908 | GEO
Project description:Tumor-associated breast vasculature was laser-cappture microdissected from IDC breast cancer cases. The goal of the study was to characterize the heterogeneity of breast tumor-associated vasculature and identify gene expression signatures predictive of clinical outcome. common reference design, 32 samples
2010-09-19 | E-GEOD-15363 | biostudies-arrayexpress
Project description:Purpose: To better characterise the population structure of primary bovine retinal microvascular endothelial cells (RMECs) based upon their individual transcriptomes. Methods: Individual RMECs were captured on the Fluidigm C1 system (Fluidigm), cDNA libraries were prepared using a Nextera XT kit and sequencing performed on a NextSeq (Illumina). Results: Application of a single cell RNA-seq analysis workflow showed that RMECs form a relatively homogeneous population in culture, with the main subgroup being proliferating cells. Expression of markers from along the arteriovenous tree suggests that most cells originate from capillaries. An in silico model of the blood retinal barrier was created, including junctional proteins not previously reported within the retinal vasculature. Numerous alternative splicing events involving exons within microvascular barrier genes were observed and in many cases individual cells expressed exclusively one isoform. Conclusions: We have optimised a workflow for single-cell transcriptomics in primary RMECs. Our results have provided fundamental insights into the genes involved in retinal microvascular barrier formation.
2020-09-01 | GSE136800 | GEO
Project description:Human herpesvirus-8 (HHV-8) is the causative agent of Kaposiâs sarcoma and is associated with the angioproliferative disorders primary effusion lymphoma (PEL) and multicentric Castlemanâs disease (MCD). We have previously described evidence of HHV-8 infection within the pulmonary vasculature of patients with idiopathic pulmonary arterial hypertension (IPAH). We speculated that viral infection of the pulmonary microvascular endothelial cells could cause the angioproliferative phenotype characteristic of severe pulmonary arterial hypertension (PAH). We now demonstrate the ability of HHV-8 to infect human pulmonary microvascular endothelial cells (HPMVECs) in vitro, confirming both latent and lytic infection. HHV-8 infection of HPMVECs resulted in significant changes of gene expression including alterations of pathways integral to both cellular apoptosis and angiogenesis. This infection also results in alterations of genes integral to the bone morphogenic protein (BMP) pathway, including down regulation of bone morphogenic protein receptor 1a (BMPR1a) and bone morphogenic protein 4 (BMP4). Other genes previously implicated in the development of PAH are also altered in expression by HHV-8 infection. These include increased expression of Interleukin-6 (IL-6) and the matrix metalloproteinases (MMP)-1, MMP-2 and MMP-10. Lastly, cells infected with HHV-8 apoptosis resistant. Infection of pulmonary microvascular endothelial cells with human herepesvirus-8 results in alteration of the BMP pathway as well as an anti-apoptotic phenotype, consistent with the development of plexiform lesions characteristic of pulmonary arterial hypertension. Experiment Overall Design: ⢠Direct comparison of HHV8-infected and mock-infected human pulmonary microvascular endothelial cells. Experiment Overall Design: ⢠Triplicate infection and mock infection samples were prepared. One hybridization per sample, 6 total hybridizations Experiment Overall Design: ⢠Single channel hybridization (no reference).
2008-06-14 | E-GEOD-6489 | biostudies-arrayexpress
Project description:A rapid and robust method for Mitochondrial DNA profiling
| PRJNA1108319 | ENA
Project description:Tumor-associated breast vasculature was laser-cappture microdissected from IDC breast cancer cases. The goal of the study was to characterize the heterogeneity of breast tumor-associated vasculature and identify gene expression signatures predictive of clinical outcome.
2010-09-19 | GSE15363 | GEO
Project description:Human herpesvirus-8 (HHV-8) is the causative agent of Kaposi’s sarcoma and is associated with the angioproliferative disorders primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD). We have previously described evidence of HHV-8 infection within the pulmonary vasculature of patients with idiopathic pulmonary arterial hypertension (IPAH). We speculated that viral infection of the pulmonary microvascular endothelial cells could cause the angioproliferative phenotype characteristic of severe pulmonary arterial hypertension (PAH). We now demonstrate the ability of HHV-8 to infect human pulmonary microvascular endothelial cells (HPMVECs) in vitro, confirming both latent and lytic infection. HHV-8 infection of HPMVECs resulted in significant changes of gene expression including alterations of pathways integral to both cellular apoptosis and angiogenesis. This infection also results in alterations of genes integral to the bone morphogenic protein (BMP) pathway, including down regulation of bone morphogenic protein receptor 1a (BMPR1a) and bone morphogenic protein 4 (BMP4). Other genes previously implicated in the development of PAH are also altered in expression by HHV-8 infection. These include increased expression of Interleukin-6 (IL-6) and the matrix metalloproteinases (MMP)-1, MMP-2 and MMP-10. Lastly, cells infected with HHV-8 apoptosis resistant. Infection of pulmonary microvascular endothelial cells with human herepesvirus-8 results in alteration of the BMP pathway as well as an anti-apoptotic phenotype, consistent with the development of plexiform lesions characteristic of pulmonary arterial hypertension. Keywords: Viral infection of endothelial cells in culture
2008-04-30 | GSE6489 | GEO